Method of Treating Dyspnea Associated with Acute Heart Failure

ABSTRACT

The disclosure pertains to methods of reducing decompensation through acute intervention including in subjects afflicted with acute decompensated heart failure. Particularly, the disclosure provides methods for treating acute cardiac decompensation by administering a pharmaceutically effective amount of relaxin.

RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application Nos. 61/164,333, filed Mar. 27, 2009,61/201,240, filed Dec. 8, 2008, 61/190,545, filed Aug. 28, 2008, and61/127,889, filed May 16, 2008, all of which are incorporated herein byreference in their entirety for all purposes.

FIELD

The present disclosure relates to methods for treating decompensation inhuman subjects afflicted with symptoms of acute decompensated heartfailure. The methods described herein employ administration of relaxin.

BACKGROUND

Acute heart failure (AHF) or acute decompensated heart failure (ADHF)encompasses a heterogeneous group of disorders that typically includesdyspnea (shortness of breath), edema (fluid retention) and fatigue. Forexample, a patient who presents with shortness of breath from anexacerbation of congestive heart failure would fall within the group ofAHF patients. However, the diagnosis of AHF can be difficult and theoptimal treatment remains poorly defined despite the high prevalence ofthis condition and its association with major morbidity and mortality.The difficulties surrounding treatment begin with the lack of a cleardefinition of the disease. The term “acute decompensated heart failure”broadly represents new or worsening symptoms or signs of dyspnea,fatigue or edema that lead to hospital admission or unscheduled medicalcare. These symptoms are consistent with an underlying worsening of leftventricular function. “Acute heart failure” is sometimes defined as theonset of symptoms or signs of heart failure in a patient with no priorhistory of heart failure and previously normal function. This is anuncommon cause of AHF, particularly in patients without concomitantacute coronary syndromes. More frequently, AHF occurs in patients withpreviously established myocardial dysfunction (systolic or diastolic)such as in congestive heart failure (CHF) patients who present with anexacerbation of symptoms or signs after a period of relative stability(Allen and O'Connor, CMAJ 176(6):797-805, 2007). Consequently, AHF canresult without prior history of CHF, be based on a pathophysiologicalorigin in prior CHF patients (functional), or be the result of anatomiccauses in prior CHF patients (structural). Thus, AHF can be a functionaland/or a structural disease.

The identification of the acute triggers for the decompensation, as wellas noninvasive characterization of cardiac filling pressures and cardiacoutput is central to management. Diuretics, vasodilators, continuouspositive airway pressure and inotropes can be used to alleviatesymptoms. However, there are no agents currently available for thetreatment of AHF that have been shown (in large prospective randomizedclinical trials) to provide significant improvements inintermediate-term clinical outcomes.

AHF is the single most costly hospital admission diagnosis according tothe Center for Medicare and Medicaid Administration. AHF accounts formore than one million hospitalizations per year and re-hospitalizationswithin six months are as high as fifty percent. The annual mortalityrate approaches fifty percent (for those patients with New York HeartAssociation class III or IV symptoms). Generally, non-aggressive medicalcare during the initial hospitalization, sub-optimal treatment beforere-admission, and patient noncompliance contribute strongly to the highreadmission rate. Fifty percent of patients with classic AHF symptomsbefore admission receive no alteration in their treatment at the initialconsultation with their health care provider (McBride et al.,Pharmacotherapy 23(8):997-1020, 2003).

While AHF was traditionally viewed as a disorder associated with sodiumand water retention and left ventricular (LV) dysfunction, it is nowalso understood to be associated with neurohormonal activation (Schrieret al., The New England Journal of Medicine 341(8):577-585, 1999). Asindicated above, the clinical syndrome of AHF is characterized by thedevelopment of dyspnea associated with the rapid accumulation of fluidwithin the lung's interstitial and alveolar spaces, resulting fromacutely elevated cardiac filling pressures (cardiogenic pulmonaryedema). More specifically, AHF can also present as elevated leftventricular filling pressures and dyspnea without pulmonary edema. It ismost commonly due to left ventricular systolic or diastolic dysfunction,with or without additional cardiac pathology, such as coronary arterydisease or valve abnormalities. In addition, a variety of conditions orevents can cause cardiogenic pulmonary edema due to an elevatedpulmonary capillary wedge pressure in the absence of heart disease,including severe hypertension, particularly renovascular hypertension,and severe renal disease.

Hospital admissions for AHF have increased during the past few decadesand are projected to continue to increase in the future. AHF is usuallydiagnosed and managed based on tradition rather than evidence. In orderto reduce the costs associated with this disorder and optimize patientoutcomes, new approaches and better treatment options are essential.Diuretic therapy has been the main treatment for symptom relief forpulmonary congestion and fluid retention. Continuous infusions of loopdiuretic therapy rather than bolus dosing may enhance efficacy andreduce the extent of diuretic resistance. Catecholamine- andphosphodiesterase-based inotropic therapies are efficacious, but theincreased risk of arrhythmogenesis and the potential for negativeeffects on survival limit their use. NATROCOR (nesiritide marketed byScios) used in vasodilator therapy, is a pharmacological preload andafterload reducer, but based on clinical trial evidence should bereserved for those with resistance to intravenous nitrate therapy(McBride et al., supra). Vasopressin receptor antagonists and adenosinereceptor antagonists offer some improved renal preservation duringaggressive diuresis (Tang et al., Current Cardiology Reviews 1(1):1-5,2005).

Volume and perfusion status provide useful clues to a patient's cardiacperformance and help shape the treatment plan for patients with AHF.Caregivers must frequently reassess the patient's hemodynamic status todetermine volume and perfusion status. Volume status is determined byassessing if the patient is wet, dry, or has a balanced fluid level(hypervolemia, hypovolemia, or euvolemia, respectively), and perfusionis assessed by determining if the patient is cold, cool/lukewarm, orwarm (has perfusion that is very low, slightly low, or normal,respectively). Evidence of congestion includes the signs of neck veindistension, elevated pressure in the right internal jugular vein,positive abdominal-jugular neck vein reflex, edema, ascites, andcrackles (rarely), as well as the symptoms of dyspnea, orthopnea, andparoxysmal nocturnal dyspnea. In addition, various tests can beperformed at the time of admission including chest radiographs, arterialblood gas levels, liver function tests, hematologic tests,electrocardiograms, and basic metabolic profile. The findings onphysical examination and the results of assays of serum levels ofnatriuretic peptides can be used to guide treatment in patients withacute decompensated heart failure. Brain natriuretic peptide or B-typenatriuretic peptide (BNP) is secreted mainly from the ventricularmyocardium in response to elevations in end-diastolic pressure andventricular volume expansion. The measurement of BNP can aid indiagnosis of CHF as AHF, and BNP levels can also be used to assessclinical status and the effectiveness of therapies during an admissionfor acute decompensation (Albert et al., Critical Care Nurse24(6):14-29, 2004).

While significant advances have been made in the realm of chronic heartfailure management, clinicians continue to grapple with optimalstrategies to treat acutely decompensated patients including patientsafflicted with AHF. There is now an increasing awareness of the complexinterplay that occurs between the heart and kidneys among patients withheart failure. As such, many of the traditional therapeutics used totreat this patient population can significantly alter renal function andare, thus, no longer considered optimal treatment options. A morecomprehensive approach is desired and the present disclosure addressesthis need.

BRIEF SUMMARY OF PREFERRED EMBODIMENTS

The present disclosure provides methods for treating conditionsassociated with acute decompensated heart failure (AHF) by administeringrelaxin. The number of hospital admissions due to AHF related symptomsare on the steady rise and the cost associated with caring for thispopulation of patients is staggering. Thus, a new therapeutic approachis needed and the disclosure addresses this need. One advantage of thisdisclosure is that the administration of relaxin results in a balancedvasodilation that prevents subjects diagnosed with conditions associatedwith AHF from further deteriorating. As such, the subjects can bemaintained at a steady-state level where hospitalization is not requiredand the number or duration of hospital visits is significantly reduced.Another advantage of the present disclosure is that relaxin, whenadministered to patients, shows effectiveness with little to no adversedrug reactions (ADRs). Herein, relaxin is shown to have a beneficialeffect on reducing acute decompensation without causing ADRs. Thus, thepresent disclosure provides a treatment that leads to balancedvasodilation in a specific patient population that suffers from acutedecompensation and is specifically suited to benefit from relaxintreatment.

One aspect of the disclosure provides a method of reducing acute cardiacdecompensation events including selecting a human subject with acutecardiac decompensation, wherein the subject has a vasculature and thevasculature has relaxin receptors. The method further includesadministering to the subject a pharmaceutical formulation includingpharmaceutically active relaxin in an amount effective to reduce acutecardiac decompensation in the subject by binding to the relaxinreceptors in the vasculature of the subject, resulting in balancedvasodilation. The cardiac decompensation can be due to any one or morecauses, including but not limited to, neurohormonal imbalance, fluidoverload, cardiac arrhythmia, and cardiac ischemia. In one embodiment,the human subject suffers from acute vascular failure.

Relaxin employed in the pharmaceutical formulations of the disclosurecan be, for example, synthetic or recombinant relaxin, or apharmaceutically effective relaxin agonist. In one embodiment of thedisclosure, relaxin is H1 human relaxin. In another embodiment, relaxinis H2 human relaxin. In yet another embodiment, relaxin is H3 humanrelaxin. In a further embodiment, relaxin is synthetic or recombinanthuman relaxin, or a pharmaceutically effective relaxin agonist. Thus,the subject can be treated with a pharmaceutical formulation ofsynthetic or recombinant human relaxin or relaxin agonist. In oneembodiment of the disclosure, the subject is treated with synthetichuman relaxin. In another embodiment, the subject is treated withrecombinant human relaxin. In yet another embodiment, the subject istreated with a pharmaceutically effective relaxin agonist. Relaxin canbe administered to the subject through a number of different routes,including but not limited to, intravenously, subcutaneously,intramuscularly, sublingually and via inhalation. More specifically, thepharmaceutical formulation of relaxin or relaxin agonist can beadministered to the subject in an amount in a range of about 10 to 1000μg/kg of subject body weight per day. As such, relaxin is administeredto the subject so as to maintain a serum concentration of relaxin offrom about 1 to 500 ng/ml.

Human subjects that would benefit from the methods of the disclosureusually present with acute cardiac decompensation events, including butnot limited to, dyspnea, hypertension, arrhythmia, reduced renal bloodflow, and renal insufficiency, wherein these events are often associatedwith readmission to the hospital. In one embodiment of the disclosure,these acute cardiac decompensation events are pathophysiological innature. Most commonly, such events are associated with acutedecompensated heart failure (AHF). In one embodiment, the human subjectsuffers from acute vascular failure. In another embodiment, the acutecardiac decompensation is intermittent. In an alternative embodiment,the acute cardiac decompensation is chronic.

Another aspect of the disclosure provides a method of treating acutecardiac decompensation associated with acute decompensated heart failure(AHF). The method includes selecting a human subject with acute cardiacdecompensation, wherein the subject has a vasculature and thevasculature has relaxin receptors, and further, administering to thesubject a pharmaceutical formulation including pharmaceutically activerelaxin or pharmaceutically effective relaxin agonist. Relaxin isadministered in an amount effective to reduce the acute cardiacdecompensation in the subject by binding to the relaxin receptors in thevasculature of the subject, resulting in balanced vasodilation. Thecardiac decompensation can be due to any one or more causes, includingbut not limited to, neurohormonal imbalance, fluid overload, cardiacarrhythmia, and cardiac ischemia. In one embodiment, the human subjectsuffers from acute vascular failure.

The disclosure further encompasses a method of treating acute cardiacdecompensation associated with acute decompensated heart failure (AHF),including administering a formulation which includes pharmaceuticallyactive synthetic human relaxin or pharmaceutically effective relaxinagonist to a human subject in an amount in a range of about 10 to 1000μg/kg of subject body weight per day, and continuing the administrationover a period of time sufficient to achieve an amelioration in acutecardiac decompensation events, including but not limited to, dyspnea,hypertension, arrhythmia, reduced renal blood flow, and renalinsufficiency. In one preferred embodiment, pharmaceutically effectiverelaxin or an agonist thereof is administered at about 30 μg/kg/daywhich results in serum concentrations of 10 ng/ml. In another preferredembodiment, pharmaceutically effective relaxin or an agonist thereof isadministered at about 10 to about 250 μg/kg/day. The amelioration maymanifest itself as a reduced number of acute cardiac decompensationevents and/or less severe acute cardiac decompensation events in thesubject. In one embodiment, the human subject suffers from acutevascular failure.

Still, another aspect of the disclosure provides a method of treatingacute decompensated heart failure (AHF) in a human subject who alsosuffers from renal insufficiency. This method includes selecting a humansubject with symptoms of acute cardiac decompensation and renalinsufficiency, wherein the subject has a systemic and renal vasculaturecomprising relaxin receptors. The method further includes administeringto the subject a pharmaceutical formulation comprising pharmaceuticallyactive relaxin or pharmaceutically effective relaxin agonist, whereinrelaxin performs a dual action by binding to the relaxin receptors inthe systemic and renal vasculature of the subject, resulting in balancedvasodilation. In one embodiment, the human subject suffers from acutevascular failure. The cardiac decompensation can be due to any one ormore causes, including but not limited to, neurohormonal imbalance,fluid overload, cardiac arrhythmia, and cardiac ischemia. The subjectmay suffer from symptoms such as dyspnea, hypertension, arrhythmia,reduced renal blood flow, and the like, wherein the symptoms arecommonly further associated with readmission to the hospital. Notably,the subject may be further experiencing elevated levels of brainnatriuretic peptide (BNP). In addition, a reversal of the acute cardiacdecompensation may occur in combination with a decrease in circulatinglevels of BNP.

Another aspect of the present disclosure provides a method of modulatingendothelin in a human subject, including selecting a human subject witha neurohormonal imbalance, wherein the subject has a vasculature and thevasculature has relaxin receptors. The method further includesadministering to the subject a pharmaceutical formulation which includespharmaceutically active relaxin or pharmaceutically effective relaxinagonist in an amount effective to reduce the neurohormonal imbalance inthe subject by binding to the relaxin receptors in the vasculature ofthe subject, resulting in balanced vasodilation. In one embodiment, thehuman subject suffers from acute vascular failure.

The disclosure further contemplates a method of reducing mortality riskin a human patient with symptoms of acute cardiac decompensation. Thismethod includes selecting a human subject with acute cardiacdecompensation, wherein the subject has a vasculature and thevasculature has relaxin receptors, and administering to the subject apharmaceutical formulation including pharmaceutically active relaxin orpharmaceutically effective relaxin agonist. The relaxin is administeredin an amount effective to reduce the acute cardiac decompensation in thesubject by binding to the relaxin receptors in the vasculature of thesubject, thereby resulting in reduced levels of brain natriureticpeptide (BNP). The reduced levels of BNP can be physically measured inorder to predict risk of mortality in the patient. Generally, thereduced levels of BNP are due to reduced cardiac stress following areduction in vascular resistance. The reduction in vascular resistanceis in turn due to the balanced vasodilation which is the result ofrelaxin binding to relaxin receptors that are found on smooth musclecells of the renal vasculature. In one embodiment, the human subjectsuffers from acute vascular failure.

Generally, the reversal of the acute cardiac decompensation in thesubjects occurs through activation of specific relaxin receptors such asthe LGR7 and LGR8 receptors. In particular, LGR7 and LGR8 receptors areactivated through the binding of relaxin or a relaxin agonist, whereinthe binding triggers the production of nitric oxide (NO) which resultsin a balanced vasodilation. These relaxin specific receptors are locatedon smooth muscle tissue of the vasculature which includes systemic andrenal vasculature.

Yet another aspect of the disclosure provides a method of reducing acutecardiac decompensation events, including selecting a human subject withacute cardiac decompensation, wherein the subject has a vasculature andthe vasculature has relaxin receptors. The method further includesadministering to the subject a pharmaceutical formulation includingpharmaceutically active relaxin or pharmaceutically effective relaxinagonist in an amount effective to reduce acute cardiac decompensation inthe subject by binding to the relaxin receptors in the vasculature ofthe subject, resulting in balanced vasodilation, wherein the relaxin isadministered to the subject so as to maintain a serum concentration ofrelaxin of equal or greater than about 3 ng/ml. The method furtherincludes administering to the subject a pharmaceutical formulationincluding pharmaceutically active relaxin or pharmaceutically effectiverelaxin agonist in an amount effective to reduce acute cardiacdecompensation in the subject by binding to the relaxin receptors in thevasculature of the subject, resulting in balanced vasodilation, whereinthe relaxin is administered to the subject so as to maintain a serumconcentration of relaxin of equal or greater than about 10 ng/ml. In oneembodiment, the human subject suffers from acute vascular failure.

Still, another aspect of the disclosure provides relaxin for use in thetreatment of acute cardiac decompensation. The acute cardiacdecompensation is commonly associated with acute decompensated heartfailure (AHF). The method includes selecting a human subject with acutecardiac decompensation, wherein the subject has a vasculature and thevasculature has relaxin receptors, and further, administering to thesubject a pharmaceutical formulation including pharmaceutically activerelaxin or pharmaceutically effective relaxin agonist. In oneembodiment, the human subject suffers from acute vascular failure.Relaxin or a relaxin agonist is administered in an amount effective toreduce the acute cardiac decompensation in the subject by binding to therelaxin receptors in the vasculature of the subject, resulting inbalanced vasodilation. The cardiac decompensation can be due to any oneor more causes, including but not limited to, neurohormonal imbalance,fluid overload, cardiac arrhythmia, and cardiac ischemia. The disclosurealso contemplates relaxin for use in reducing acute cardiacdecompensation events.

The disclosure further encompasses relaxin for use in treating acutedecompensated heart failure (AHF) in a human subject who also suffersfrom renal insufficiency; relaxin for use in modulating endothelin in ahuman subject; and relaxin for use in reducing mortality risk in a humanpatient with symptoms of acute cardiac decompensation as discussedherein.

Another aspect of the disclosure provides a method of reducing acutecardiac decompensation events. The method includes selecting a humansubject with acute cardiac decompensation, wherein the subject has avasculature and the vasculature has relaxin receptors; and administeringto the subject a pharmaceutical formulation including pharmaceuticallyactive relaxin in an amount effective to reduce acute cardiacdecompensation in the subject by binding to the relaxin receptors in thevasculature of the subject. In this method, treatment with relaxinresults in a reduction of acute cardiac decompensation events lastingfor at least about 1 to 14 days from onset of relaxin treatment. Theacute cardiac decompensation events include, but are not limited todyspnea, extra body weight due to retention of fluids, length ofhospital stay, likelihood of hospital re-admission, need for loopdiuretics, need for intravenous nitroglycerin, and an incidence ofworsening heart failure. In one embodiment, the patients are treatedwith relaxin for 48 hours. In another embodiment, the patients aretreated with relaxin for 24 hours. In yet another embodiment, thepatients are treated with relaxin for 12 hours. In still anotherembodiment, the patients are treated with relaxin for 6 hours. Theeffects of relaxin can be measured at any time point, for example, at 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days or later.

In one preferred embodiment, relaxin is administered at about 30mcg/kg/day. In one preferred embodiment, relaxin is administered atabout 30 mcg/kg/day. In another preferred embodiment, relaxin isadministered at about 35 mcg/kg/day. In another preferred embodiment,relaxin is administered at about 40 mcg/kg/day. In another preferredembodiment, relaxin is administered at about 45 mcg/kg/day. In anotherpreferred embodiment, relaxin is administered at about 50 mcg/kg/day. Inanother preferred embodiment, relaxin is administered at about 55mcg/kg/day. In another preferred embodiment, relaxin is administered atabout 60 mcg/kg/day. In another preferred embodiment, relaxin isadministered at about 65 mcg/kg/day. In another preferred embodiment,relaxin is administered at about 70 mcg/kg/day. In another preferredembodiment, relaxin is administered at about 75 mcg/kg/day. In anotherpreferred embodiment, relaxin is administered at about 80 mcg/kg/day. Inanother preferred embodiment, relaxin is administered at about 85mcg/kg/day. In another preferred embodiment, relaxin is administered atabout 100 mcg/kg/day. Relaxin may also be administered at a dosage of 90to 200 mcg/kg/day. Pharmaceutically effective relaxin includesrecombinant or synthetic H1 human relaxin, H2 human relaxin or H3 humanrelaxin or an agonist or a variant thereof. In one preferred embodiment,relaxin is administered to the subject so as to maintain a serumconcentration of about 10 ng/ml. The pharmaceutical formulation ofrelaxin can be administered intravenously, subcutaneously,intramuscularly, sublingually or via inhalation. In one preferredembodiment, the pharmaceutical formulation of relaxin is administeredintravenously. The relaxin receptors are activated through the bindingof relaxin and include, but are not limited to, LRG7, LGR8, GPCR135, andGPCR142. The binding of relaxin to the relaxin receptors triggers theproduction of nitric oxide (NO) which results in balanced vasodilation.The relaxin receptors are located, for example, on the smooth muscletissue of the vasculature.

The present disclosure also provides a method for treating acardiovascular condition comprising: administering to a human subject apharmaceutically active H2 relaxin in an amount effective to treat thecardiovascular condition, wherein the cardiovascular condition isdiagnosed based on the presence of two or more of the group consistingof dyspnea at rest or with minimal exertion, pulmonary congestion onchest X-ray, and elevated natriuretic peptide levels [brain natriureticpeptide (BNP)≧350 pg/mL or NT-pro-BNP≧1400 pg/mL]. In some embodiments,the cardiovascular condition is acute heart failure and the two or morecomprise dyspnea at rest or with minimal exertion, and pulmonarycongestion on chest X-ray. In some embodiments, the cardiovascularcondition is acute heart failure and the two or more comprise dyspnea atrest or with minimal exertion, and elevated natriuretic peptide levels[brain natriuretic peptide (BNP)≧350 pg/mL or NT-pro-BNP≧1400 pg/mL]. Insome embodiments, the cardiovascular condition is acute heart failureand the two or more comprise pulmonary congestion on chest X-ray andelevated natriuretic peptide levels [brain natriuretic peptide (BNP)≧350pg/mL or NT-pro-BNP ≧1400 pg/mL]. In some preferred embodiments, thesubject is a male or a nonpregnant female. In some preferredembodiments, the subject has a systolic blood pressure of at least about125 mmHg.

In addition, the present disclosure provides a method for treatingdyspnea associated with acute heart failure, comprising: administeringto a human subject a pharmaceutically active H2 relaxin in an amounteffective to reduce dyspnea in the subject, wherein the subject hasdyspnea associated with acute heart failure and is in a hypertensive ornormotensive state at the onset of the administering. In someembodiments, the methods further comprise selecting the human subjecthaving dyspnea associated with acute heart failure and in a hypertensiveor normotensive state, prior to the administering step. In someembodiments, the H2 relaxin is administered for at least 24 or 48 hours,while in others the H2 relaxin is administered over 48 hours. In someembodiments, the H2 relaxin is administered at an intravenous infusionrate in the range of about 10 μg/kg/day to about 250 μg/kg/day, in arange of about 30 μg/kg/day to about 100 μg/kg/day, or at about 30μg/kg/day. In some embodiments, the reduction in dyspnea isstatistically significant at 6 hours after the onset of treatmentcompared to treatment without H2 relaxin, at 12 hours after the onset oftreatment compared to treatment without H2 relaxin, or at 6, 12 and 24hours after the onset of treatment compared to placebo. In someembodiments, the reduction in dyspnea lasts for at least about twice theduration of treatment, at least about 4 times the duration of treatment,or at least about 7 times the duration of treatment. In someembodiments, the methods further comprise reducing the body weight ofthe subject by at least about 0.5 kg over a 14-day period compared totreatment without H2 relaxin, or at least about 1 kg over a 14-dayperiod compared to treatment without H2 relaxin. In some embodiments,the subject is renally impaired. In a subset of these embodiments, thesubject has a creatinine clearance in the range of about 35 to about 75mL/min. In some embodiments, the methods further comprise reducing the60-day risk of death or rehospitalization of the subject compared totreatment of acute decompensated heart failure without H2 relaxin. In asubset of these embodiments, the 60-day risk of death orrehospitalization is reduced by at least 50%. In some preferredembodiments, the subject has dyspnea requiring hospitalization. In someembodiments, the methods further comprise reducing the hospitalizationlength of stay by at least one day compared to treatment of acutedecompensated heart failure without H2 relaxin. In some methods, the H2relaxin is administered at an intravenous infusion rate in the range ofabout 30 μg/kg/day and the hospitalization length of stay is reduced byat least two days compared to treatment of acute decompensated heartfailure without H2 relaxin. In some embodiments, the methods furthercomprise reducing the 60-day risk of rehospitalization due to heartfailure or renal insufficiency of the subject compared to treatment ofacute decompensated heart failure without H2 relaxin. In some preferredembodiments, the 60-day risk of rehospitalization due to heart failureor renal insufficiency is reduced by at least about 50%. In somemethods, the H2 relaxin is administered at an intravenous infusion ratein the range of about 30 μg/kg/day and the 60-day risk ofrehospitalization due to heart failure or renal insufficiency is reducedby at least about 70%. In some embodiments, the methods comprisereducing the 180-day risk of cardiovascular death of the subjectcompared to treatment of acute decompensated heart failure without H2relaxin. In some preferred embodiments, the 180-day risk ofcardiovascular death is reduced by at least about 50%. In someembodiments, the H2 relaxin is administered at an intravenous infusionrate less than about 250 μg/kg/day and the 180-day risk ofcardiovascular death is reduced by at least about 70%. In someembodiments, the methods further comprise reducing the 180-day risk ofall-cause mortality of the subject compared to treatment of acutedecompensated heart failure without H2 relaxin. In some preferredembodiments, the 180-day risk of all-cause mortality is reduced by atleast about 25%. In some embodiments, the H2 relaxin is administered atan intravenous infusion rate less than about 250 μg/kg/day and the180-day risk of all-cause mortality is reduced by at least about 50%. Insome preferred embodiments, the subject is a male or a nonpregnantfemale. In some preferred embodiments, the subject has a systolic bloodpressure of at least about 125 mmHg.

The disclosure further provides a method for treating dyspnea associatedwith acute decompensated heart failure, comprising: administering to ahuman subject a pharmaceutically active H2 relaxin in an amounteffective to reduce dyspnea in the subject, wherein the subject hasdyspnea associated with acute decompensated heart failure and at leastone indicia of cardiac ischemia. In some embodiments, the method furthercomprises selecting the human subject having dyspnea associated withacute decompensated heart failure and at least one indicia of cardiacischemia, prior to the administering step. In some embodiments, the atleast one indicia of cardiac ischemia is selected from the groupconsisting of a positive troponin test, an abnormal electrocardiogram,the presence of chest pain, the presence of an arrhythmia, a positivecreatine kinase-MB test, and an abnormal echocardiogram. In someembodiments of the method, the subject also has a left ventricularejection fraction in the range of 20-40%. In another embodiment, thesubject has a left ventricular ejection fraction of at least 40%. Insome embodiments of the method, the subject is normotensive orhypertensive. In another embodiment, the subject has a systolic bloodpressure of at least about 125 mm Hg. In some embodiments of the method,the subject is renally impaired. In another embodiment, the subject hasa creatinine clearance in the range of about 35 to about 75 mL/min. Insome embodiments of the method for treating a cardiovascular condition,the H2 relaxin is administered for at least 24 or 48 hours. In anotherembodiment, the H2 relaxin is administered over 48 hours. In yet anotherembodiment, the H2 relaxin is administered at an infusion rate in therange of about 10 μg/kg/day to about 960 μg/kg/day. In yet anotherembodiment of the method, the H2 relaxin is administered at anintravenous infusion rate in the range of about 10 μg/kg/day to about250 μg/kg/day. In yet another embodiment, the H2 relaxin is administeredat an intravenous infusion rate in the range of about 30 μg/kg/day toabout 100 μg/kg/day. In yet another embodiment, the H2 relaxin isadministered at an intravenous infusion rate in the range of about 30μg/kg/day. In some embodiments of the method, the subject has dyspnearequiring hospitalization. In some preferred embodiments, the subject isa male or a nonpregnant female.

The disclosure further provides a method for treating acutedecompensated heart failure, comprising: a) identifying a subject withacute decompensated heart failure; b) assessing the orthopnea status inthe subject; c) selecting an initial dosage of a pharmaceutically activeH2 relaxin based upon the orthopnea status in the patient; and d)administering the dosage to the subject. In some embodiments of themethod, the selected initial dosage is higher in the presence oforthopnea than in the absence of orthopnea. In some embodiments, theselected initial dosage is at least about 30 μg/kg/day but below about100 μg/kg/day in the presence of orthopnea. In some preferredembodiments, the subject is a male or a nonpregnant female. In somepreferred embodiments, the subject has a systolic blood pressure of atleast about 125 mmHg.

The disclosure further provides a method for treating dyspnea associatedwith acute decompensated heart failure, comprising: administering to ahuman subject a pharmaceutically active H2 relaxin in an amounteffective to reduce dyspnea in the subject, wherein the subject hasacute decompensated heart failure and a left ventricular ejection fromof at least 20%. In some embodiments, the method further comprisesselecting the human subject having acute decompensated heart failure anda left ventricular ejection from of at least 20%, prior to theadministering step. In some embodiments, the subject has a leftventricular ejection fraction of at least about 20%. In some embodimentsof the method, the subject has a left ventricular ejection fraction ofat least about 40%. In one embodiment, the subject is normotensive,while in other embodiments the subject is hypertensive. In someembodiments, the subject has a systolic blood pressure of at least about125 mm Hg. In another embodiment, the subject is renally impaired. Inanother embodiment, the subject has a creatinine clearance in the rangeof about 35 to about 75 mL/min. In another embodiment of the method, theH2 relaxin is administered for at least 24 or 48 hours. In yet anotherembodiment, the H2 relaxin is administered over 48 hours. In yet anotherembodiment, the H2 relaxin is administered at an infusion rate in therange of about 10 μg/kg/day to about 960 μg/kg/day. In yet anotherembodiment, H2 relaxin is administered at an intravenous infusion ratein the range of about 10 μg/kg/day to about 250 μg/kg/day. In yetanother embodiment, H2 relaxin is administered at an intravenousinfusion rate in the range of about 30 μg/kg/day to about 100 μg/kg/day.In yet another embodiment, H2 relaxin is administered at an intravenousinfusion rate in the range of about 30 μg/kg/day. In some embodiments,the methods further comprise reducing the 60-day risk of death orrehospitalization of the subject compared to treatment of acutedecompensated heart failure without H2 relaxin. In some embodiments, the60-day risk of death or rehospitalization is reduced by at least 50%. Insome embodiments of the method, the subject has dyspnea requiringhospitalization. In some embodiments, the methods further comprisereducing the hospitalization length of stay by at least one day comparedto treatment of acute decompensated heart failure without H2 relaxin. Insome embodiments, the H2 relaxin is administered at an intravenousinfusion rate in the range of about 30 μg/kg/day and the hospitalizationlength of stay is reduced by at least two days compared to treatment ofacute decompensated heart failure without H2 relaxin. In someembodiments, the methods further comprise reducing the 60-day risk ofrehospitalization due to heart failure or renal insufficiency of thesubject compared to treatment of acute decompensated heart failurewithout H2 relaxin. In another embodiment, the 60-day risk ofrehospitalization due to heart failure or renal insufficiency is reducedby at least about 50%. In another embodiment, the H2 relaxin isadministered at an intravenous infusion rate in the range of about 30μg/kg/day and the 60-day risk of rehospitalization due to heart failureor renal insufficiency is reduced by at least about 70%. In someembodiments, the methods further comprise reducing the 180-day risk ofcardiovascular death of the subject compared to treatment of acutedecompensated heart failure without H2 relaxin. In some embodiments, the180-day risk of cardiovascular death is reduced by at least about 50%.In some embodiments of the method, the H2 relaxin is administered at anintravenous infusion rate less than about 250 μg/kg/day and the 180-dayrisk of cardiovascular death is reduced by at least about 70%. In someembodiments, the methods further comprise reducing the 180-day risk ofall-cause mortality of the subject compared to treatment of acutedecompensated heart failure without H2 relaxin. In another embodiment,the 180-day risk of all-cause mortality is reduced by at least about25%. In another embodiment, the H2 relaxin is administered at anintravenous infusion rate less than about 250 μg/kg/day and the 180-dayrisk of all-cause mortality is reduced by at least about 50%. In somepreferred embodiments, the subject is a male or a nonpregnant female.

The disclosure further provides a method for treating acutedecompensated heart failure, comprising: a) selecting a subject withacute decompensated heart failure and a systolic blood pressure of atleast 125 mm Hg; and b) administering to the subject a pharmaceuticallyactive H2 relaxin in an amount effective to reduce in-hospital worseningheart failure in the subject. In some embodiments of the method, thesubject is renally impaired. In some preferred embodiments, thein-hospital worsening heart failure comprises one or more of worseningdyspnea, need for additional intravenous therapy to treat the heartfailure, need for mechanical support of breathing, and need formechanical support of blood pressure. In another embodiment, the subjecthas a creatinine clearance in the range of about 35 to about 75 mL/min.In some embodiments, the H2 relaxin is administered for at least 24 or48 hours. In some embodiments, the H2 relaxin is administered over 48hours. In another embodiment, the H2 relaxin is administered at aninfusion rate in the range of about 10 μg/kg/day to about 960 μg/kg/day.In another embodiment, the H2 relaxin is administered at an intravenousinfusion rate in the range of about 10 μg/kg/day to about 250 μg/kg/day.In yet another embodiment, the H2 relaxin is administered at anintravenous infusion rate in the range of about 30 μg/kg/day to about100 μg/kg/day. In yet another embodiment, the H2 relaxin is administeredat an intravenous infusion rate in the range of about 30 μg/kg/day. Insome embodiments, the method further comprises reducing the 60-day riskof death or rehospitalization of the subject compared to treatment ofacute decompensated heart failure without H2 relaxin. In someembodiments, the 60-day risk of death or rehospitalization is reduced byat least 50%. In some embodiments, the subject has pulmonary congestionas defined by the presence of interstitial edema on chest radiograph. Insome embodiments, the method further comprises reducing thehospitalization length of stay by at least one day compared to treatmentof acute decompensated heart failure without H2 relaxin. In someembodiments, the H2 relaxin is administered at an intravenous infusionrate in the range of about 30 μg/kg/day and the hospitalization lengthof stay is reduced by at least two days compared to treatment of acutedecompensated heart failure without H2 relaxin. In some embodiments, themethod further comprises reducing the 60-day risk of rehospitalizationdue to heart failure or renal insufficiency of the subject compared totreatment of acute decompensated heart failure without H2 relaxin. Insome embodiments, the 60-day risk of rehospitalization due to heartfailure or renal insufficiency is reduced by at least about 50%. In someembodiments, the H2 relaxin is administered at an intravenous infusionrate in the range of about 30 μg/kg/day and the 60-day risk ofrehospitalization due to heart failure or renal insufficiency is reducedby at least about 70%. In some embodiments, the method further comprisesreducing the 180-day risk of cardiovascular death of the subjectcompared to treatment of acute decompensated heart failure without H2relaxin. In another embodiment, the 180-day risk of cardiovascular deathis reduced by at least about 50%. In another embodiment, the H2 relaxinis administered at an intravenous infusion rate less than about 250μg/kg/day and the 180-day risk of cardiovascular death is reduced by atleast about 70%. In some embodiments, the method further comprisesreducing the 180-day risk of all-cause mortality of the subject comparedto treatment of acute decompensated heart failure without H2 relaxin. Inanother embodiment of the method, the 180-day risk of all-causemortality is reduced by at least about 25%. In another embodiment, theH2 relaxin is administered at an intravenous infusion rate less thanabout 250 μg/kg/day and the 180-day risk of all-cause mortality isreduced by at least about 50%. In some preferred embodiments, thesubject is a male or a nonpregnant female.

The disclosure further provides a method for treating, acutedecompensated heart failure comprising: a) selecting a subject withacute decompensated heart failure and a left ventricular ejectionfraction of at least about 20%; and b) administering to the subject apharmaceutically active H2 relaxin in an amount effective to reduce atleast one acute heart failure sign or symptom in the subject. In someembodiments, the at least one acute heart failure sign or symptomcomprises one or more of the group consisting of dyspnea at rest,orthopnea, dyspnea on exertion, edema, rales, pulmonary congestion,jugular venous pulse or distension, edema associated weight gain, highpulmonary capillary wedge pressure, high left ventricular end-diastolicpressure, high systemic vascular resistance, low cardiac output, lowleft ventricular ejection fraction, need for intravenous diuretictherapy, need for additional intravenous vasodilator therapy, andincidence of worsening in-hospital heart failure. In another embodiment,the subject has a left ventricular ejection fraction of at least 40%. Inanother embodiment, the subject is normotensive or hypertensive. In yetanother embodiment, the subject has a systolic blood pressure of atleast about 125 mm Hg. In some embodiments, the subject is renallyimpaired. In another embodiment, the subject has a creatinine clearancein the range of about 35 to about 75 mL/min. In some embodiments of themethod, the H2 relaxin is administered for at least 24 or 48 hours. Inanother embodiment, the H2 relaxin is administered over 48 hours. Inanother embodiment, the H2 relaxin is administered at an infusion ratein the range of about 10 μg/kg/day to about 960 μg/kg/day. In yetanother embodiment, the H2 relaxin is administered at an intravenousinfusion rate in the range of about 10 μg/kg/day to about 250 μg/kg/day.In yet another embodiment, the H2 relaxin is administered at anintravenous infusion rate in the range of about 30 μg/kg/day to about100 μg/kg/day. In yet another embodiment, the H2 relaxin is administeredat an intravenous infusion rate in the range of about 30 μg/kg/day. Insome embodiments, the method further comprises reducing the 60-day riskof death or rehospitalization of the subject compared to treatment ofacute decompensated heart failure without H2 relaxin. In someembodiments, the 60-day risk of death or rehospitalization is reduced byat least 50%. In some embodiments, the subject has dyspnea requiringhospitalization. In some embodiments, the method further comprisesreducing the hospitalization length of stay by at least one day comparedto treatment of acute decompensated heart failure without H2 relaxin. Inanother embodiment, the H2 relaxin is administered at an intravenousinfusion rate in the range of about 30 μg/kg/day and the hospitalizationlength of stay is reduced by at least two days compared to treatment ofacute decompensated heart failure without H2 relaxin. In someembodiments, the method further comprises reducing the 60-day risk ofrehospitalization due to heart failure or renal insufficiency of thesubject compared to treatment of acute decompensated heart failurewithout H2 relaxin. In another embodiment, the 60-day risk ofrehospitalization due to heart failure or renal insufficiency is reducedby at least about 50%. In another embodiment, the H2 relaxin isadministered at an intravenous infusion rate in the range of about 30μg/kg/day and the 60-day risk of rehospitalization due to heart failureor renal insufficiency is reduced by at least about 70%. In someembodiments, the method further comprises reducing the 180-day risk ofcardiovascular death of the subject compared to treatment of acutedecompensated heart failure without H2 relaxin. In some embodiments, the180-day risk of cardiovascular death is reduced by at least about 50%.In another embodiment, the H2 relaxin is administered at an intravenousinfusion rate less than about 250 μg/kg/day and the 180-day risk ofcardiovascular death is reduced by at least about 70%. In someembodiments, the method further comprises reducing the 180-day risk ofall-cause mortality of the subject compared to treatment of acutedecompensated heart failure without H2 relaxin. In another embodiment,the 180-day risk of all-cause mortality is reduced by at least about25%. In another embodiment, the H2 relaxin is administered at anintravenous infusion rate less than about 250 μg/kg/day and the 180-dayrisk of all-cause mortality is reduced by at least about 50%. In somepreferred embodiments, the subject is a male or a nonpregnant female.

The disclosure further provides a method for treating acutedecompensated heart failure, comprising administering to a subject withacute decompensated heart failure a pharmaceutically active H2 relaxinin an amount effective to reduce diuretic use during a hospital staycompared to treatment of acute decompensated heart failure without usingH2 relaxin. In some embodiments, the H2 relaxin is administered at aninfusion rate in the range of about 10 μg/kg/day to about 100 μg/kg/day.In some embodiments, the loop diuretic use during the hospital stay isreduced compared to treatment of acute decompensated heart failurewithout H2 relaxin. In another embodiment, the loop diuretic use isreduced by at least 10% over a 14-day period compared to treatmentwithout H2 relaxin. In yet another embodiment, the loop diuretic use isreduced by at least 20% over a 14-day period compared to treatmentwithout H2 relaxin. In yet another embodiment, the loop diuretic use isreduced by at least 30% over a 14-day period compared to treatmentwithout H2 relaxin. In some embodiments, the subject has a leftventricular ejection fraction of at least 40%. In some embodiments, thesubject is normotensive or hypertensive. In another embodiment, thesubject has a systolic blood pressure of at least about 125 mm Hg. Inanother embodiment, the subject is renally impaired. In yet anotherembodiment, the subject has a creatinine clearance in the range of about35 to about 75 mL/min. In some embodiments, the H2 relaxin isadministered for at least 24 or 48 hours. In another embodiment, the H2relaxin is administered over 48 hours. In yet another embodiment, the H2relaxin is administered at an infusion rate in the range of about 10μg/kg/day to about 960 μg/kg/day. In yet another embodiment, the H2relaxin is administered at an intravenous infusion rate in the range ofabout 10 μg/kg/day to about 250 μg/kg/day. In yet another embodiment,the H2 relaxin is administered at an intravenous infusion rate in therange of about 30 μg/kg/day to about 100 μg/kg/day. In yet anotherembodiment, the H2 relaxin is administered at an intravenous infusionrate in the range of about 30 μg/kg/day. In some embodiments, the methodfurther comprises reducing the 60-day risk of death or rehospitalizationof the subject compared to treatment of acute decompensated heartfailure without H2 relaxin. In some embodiments, the 60-day risk ofdeath or rehospitalization is reduced by at least 50%. In someembodiments, the subject has dyspnea requiring hospitalization. In someembodiments, the method further comprises reducing the hospitalizationlength of stay by at least one day compared to treatment of acutedecompensated heart failure without H2 relaxin. In some embodiments, theH2 relaxin is administered at an intravenous infusion rate in the rangeof about 30 μg/kg/day and the hospitalization length of stay is reducedby at least two days compared to treatment of acute decompensated heartfailure without H2 relaxin. In some embodiments, the method furthercomprises reducing the 60-day risk of rehospitalization due to heartfailure or renal insufficiency of the subject compared to treatment ofacute decompensated heart failure without H2 relaxin. In someembodiments, the 60-day risk of rehospitalization due to heart failureor renal insufficiency is reduced by at least about 50%. In anotherembodiment, the H2 relaxin is administered at an intravenous infusionrate in the range of about 30 μg/kg/day and the 60-day risk ofrehospitalization due to heart failure or renal insufficiency is reducedby at least about 70%. In some embodiments, the method further comprisesreducing the 180-day risk of cardiovascular death of the subjectcompared to treatment of acute decompensated heart failure without H2relaxin. In some embodiments, the 180-day risk of cardiovascular deathis reduced by at least about 50%. In another embodiment, the H2 relaxinis administered at an intravenous infusion rate less than about 250μg/kg/day and the 180-day risk of cardiovascular death is reduced by atleast about 70%. In some embodiments, the method further comprisesreducing the 180-day risk of all-cause mortality of the subject comparedto treatment of acute decompensated heart failure without H2 relaxin. Insome embodiments, the 180-day risk of all-cause mortality is reduced byat least about 25%. In another embodiment, the H2 relaxin isadministered at an intravenous infusion rate less than about 250μg/kg/day and the 180-day risk of all-cause mortality is reduced by atleast about 50%. In some preferred embodiments, the subject is a male ora nonpregnant female. In some preferred embodiments, the subject has asystolic blood pressure of at least about 125 mmHg. Moreover thedisclosure provides a method for treating acute decompensated heartfailure, comprising administering to a human subject with acutedecompensated heart failure a pharmaceutically active H2 relaxin in anamount effective to reduce the 60-day risk of death or rehospitalizationof the subject compared to treatment of acute decompensated heartfailure without H2 relaxin. In some embodiments, the subject has atleast one acute heart failure sign or symptom selected from the groupconsisting of dyspnea at rest, orthopnea, dyspnea on exertion, edema,rales, pulmonary congestion, jugular venous pulse or distension, edemaassociated weight gain, high pulmonary capillary wedge pressure, highleft ventricular end-diastolic pressure, high systemic vascularresistance, low cardiac output, low left ventricular ejection fraction,need for intravenous diuretic therapy, need for additional intravenousvasodilator therapy, and incidence of worsening in-hospital heartfailure. In another embodiment, the subject has a left ventricularejection fraction of at least 20% or at least 40%. In anotherembodiment, the subject is normotensive or hypertensive. In yet anotherembodiment, the subject has a systolic blood pressure of at least about125 mm Hg. In some embodiments, the subject is renally impaired. Inanother embodiment, the subject has a creatinine clearance in the rangeof about 35 to about 75 mL/min. In some embodiments of the method, theH2 relaxin is administered for at least 24 hours. In another embodiment,the H2 relaxin is administered over 48 hours. In another embodiment, theH2 relaxin is administered at an infusion rate in the range of about 10μg/kg/day to about 960 μg/kg/day. In yet another embodiment, the H2relaxin is administered at an intravenous infusion rate in the range ofabout 10 μg/kg/day to about 250 μg/kg/day. In yet another embodiment,the H2 relaxin is administered at an intravenous infusion rate in therange of about 30 μg/kg/day to about 100 μg/kg/day. In yet anotherembodiment, the H2 relaxin is administered at an intravenous infusionrate in the range of about 30 μg/kg/day. In some embodiments, the 60-dayrisk of death or rehospitalization is reduced by at least 50%. In someembodiments, the subject has dyspnea requiring hospitalization. In someembodiments, the method further comprises reducing the hospitalizationlength of stay by at least one day compared to treatment of acutedecompensated heart failure without H2 relaxin. In another embodiment,the H2 relaxin is administered at an intravenous infusion rate in therange of about 30 μg/kg/day and the hospitalization length of stay isreduced by at least two days compared to treatment of acutedecompensated heart failure without H2 relaxin. In some embodiments, themethod further comprises reducing the 60-day risk of rehospitalizationdue to heart failure or renal insufficiency of the subject compared totreatment of acute decompensated heart failure without H2 relaxin. Inanother embodiment, the 60-day risk of rehospitalization due to heartfailure or renal insufficiency is reduced by at least about 50%. Inanother embodiment, the H2 relaxin is administered at an intravenousinfusion rate in the range of about 30 μg/kg/day and the 60-day risk ofrehospitalization due to heart failure or renal insufficiency is reducedby at least about 70%. In some embodiments, the method further comprisesreducing the 180-day risk of cardiovascular death of the subjectcompared to treatment of acute decompensated heart failure without H2relaxin. In some embodiments, the 180-day risk of cardiovascular deathis reduced by at least about 50%. In another embodiment, the H2 relaxinis administered at an intravenous infusion rate less than about 250μg/kg/day and the 180-day risk of cardiovascular death is reduced by atleast about 70%. In some embodiments, the method further comprisesreducing the 180-day risk of all-cause mortality of the subject comparedto treatment of acute decompensated heart failure without H2 relaxin. Inanother embodiment, the 180-day risk of all-cause mortality is reducedby at least about 25%. In another embodiment, the H2 relaxin isadministered at an intravenous infusion rate less than about 250μg/kg/day and the 180-day risk of all-cause mortality is reduced by atleast about 50%. In some preferred embodiments, the subject is a male ora nonpregnant female.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is best understood when read in conjunction withthe accompanying figures, which serve to illustrate the preferredembodiments. It is understood, however, that the disclosure is notlimited to the specific embodiments disclosed in the figures.

FIG. 1A depicts the peptide hormone H2 relaxin which is similar in sizeand shape to insulin. FIG. 1B provides the amino acid sequence of the Bchain (SEQ ID NO:1) and the A chain (SEQ ID NO:2 with X representingglutamic acid [E] or glutamine [Q]) of human relaxin 2 (H2).

FIG. 2 is an illustration of a possible mechanism of action for relaxin.Relaxin receptors LGR7 and LGR8 bind relaxin which activates matrixmetalloproteinases MMP-2 and MMP-9 to convert endothelin-1 to truncatedendothelin-1 (1-32) which in turn binds to the endothelin B receptor(ET_(B) receptor). This triggers nitric oxide synthase (NOS) to producenitric oxide (NO) which increases vasodilation.

FIG. 3 is an illustration of the lumen of a blood vessel. Arrows showthe smooth muscle cells (SM) and the endothelium (E). Relaxin receptorsare located on the smooth muscle cells of the blood vessels (systemicand renal vasculature).

FIG. 4 depicts stable decreases in systolic blood pressure (SBP) inhypertensive and normotensive subjects in the clinical trial of relaxinin patients with systemic sclerosis. Decreases in blood pressure inpatients that were hypertensive at study entry was greater than thedecreases in blood pressure in patients that were normotensive at studyentry. Blood pressure decreases were stable during the six months ofcontinuous dosing. None of the patients developed hypotension duringdosing.

FIG. 5 depicts stable improvement in renal function, measured aspredicted creatinine clearance (CrCl), during six months of continuousdosing with relaxin but not with placebo in patients with systemicsclerosis.

FIG. 6 depicts a Likert graph of percent moderate or marked improvementin dyspnea in AHF patients treated with various dosages of relaxin(i.e., 10, 30, 100 and 250 μg/kg/day). as an average of all time points.

FIG. 7 depicts a Likert graph of percent moderate or marked improvementin dyspnea when patients suffering from AHF with a systolic bloodpressure (SBP) greater than the median were treated with various dosagesof relaxin (i.e., 10, 30, 100 and 250 μg/kg/day). A beneficial effectwas first seen at 6 hours of treatment and relaxin administered at 30μg/kg/day showed a sustained effect with about 90% improvement lastingover a period of 14 days. In comparison, placebo treated patientscontinued to decline after the placebo effect wore off.

FIG. 8 depicts a Likert graph of percent moderate or marked improvementin dyspnea when patients suffering from AHF with a creatinine clearance(CrCl) of less than the median were treated with various dosages ofrelaxin (i.e., 10, 30, 100 and 250 μg/kg/day) over a period of 48 hours.A beneficial effect was first seen at 6 hours of treatment and relaxinshowed a sustained effect across various dosages lasting over a periodof 14 days. In comparison, placebo treated patients continued to declineafter the placebo effect wore off.

FIG. 9 shows a VAS graph of dyspnea improvement when AHF patients withNT-pro-BNP levels greater than 2000 were treated with various dosages ofrelaxin (i.e., 10, 30, 100 and 250 μg/kg/day) over a period of 48 hours.A marked improvement was seen in patients treated with relaxin dosagesof 30 μg/kg/day and higher compared to patients treated with placebo.

FIG. 10 shows a VAS graph of dyspnea improvement when AHF patients withsystolic blood pressure (SBP) levels greater than the median weretreated with various dosages of relaxin (i.e., 10, 30, 100 and 250μg/kg/day) over a period of 48 hours. A particularly marked improvementwas seen in patients treated with relaxin at 30 μg/kg/day compared topatients treated with placebo.

FIG. 11 shows a VAS graph of dyspnea improvement when AHF patients withcreatinine clearance (CrCl) less than the median were treated withvarious dosages of relaxin (i.e., 10, 30, 100 and 250 μg/kg/day) over aperiod of 48 hours. A marked improvement was seen in patients treatedwith various relaxin dosages. At 30 μg/kg/day of relaxin patientsexperienced a sustained beneficial effect compared to patients treatedwith placebo.

FIG. 12 depicts a graph showing that relaxin treatment caused rapidrelief of dyspnea in AHF patients within 6, 12 and 24 hours ofadministration. In particular administration of 30 μg/kg/day of rhRlxresulted in a statistically significant improvement in dyspnea.

FIG. 13 depicts a graph showing that relaxin treatment caused sustainedrelief of dyspnea in AHF patients that lasted up to 14 days (i.e., themaximum period measured).

FIG. 14 depicts a graph showing that the placebo-treated patient groupexperienced a worsening of acute heart failure compared to therelaxin-treated groups.

FIG. 15 shows that more AHF patients in the placebo group received IVnitroglycerin by study day 5, than AHF patients in the relaxin-treatedgroups. Nitroglycerin administration is a hospital measure in theclinical study described herein.

FIGS. 16A and 16B respectively show that AHF patients in several of therelaxin treated groups had a greater reduction in body weight reflectingdiuresis, while receiving less diuretic (e.g., hospital measures andendpoints). This outcome indicates that relaxin treatment resulted inrenal vasodilation.

FIGS. 17A and 17B respectively show that relaxin treatment wasassociated with a reduction in the length of hospital stay and anincrease in longevity out of the hospital.

FIG. 18 depicts a graph shows the percent cardiovascular death (CV) orrehospitalization on day 60 in AHF patients treated with relaxin ascompared to AHF patients treated with placebo. A lower proportion ofpatients treated with relaxin had died as a result of worsenedcardiovascular disease. Likewise a lower proportion of patients treatedwith relaxin required re-hospitalization.

FIGS. 19A and 19B respectively show the percent cardiovascular (CV)death and all cause mortality in relaxin-treated AHF patients ascompared to placebo-treated AHF patients within a 180 day time framepost treatment. As illustrated in the graphs, relaxin-treated patientsfared dramatically better with a significant reduction in both thenumber of cardiovascular related deaths and in death by all causes ascompared to patients receiving placebo.

FIG. 20 shows the mean change in pulse from baseline in relaxin andplacebo treated AHF patients through day 14. The differences between thegroups are not significant, with all groups seeing a small reduction inpulse after hospital admission, indicating that relaxin treatment wasnot chronotropic.

FIG. 21 shows the mean change in systolic blood pressure (mmHg) frombaseline in relaxin and placebo treated AHF patients during infusion.The average decrease in blood pressure over all time points did notdiffer between any of the treatment groups and the placebo groups.

FIGS. 22A and 22B show that relaxin treatment reduces blood pressure inAHF patients in the study having a baseline systolic blood pressure(SBP) above the median of the group, but not in AHF patients having abaseline SBP below the median of the group. This indicates that relaxintreatment preferentially vasodilates vasoconstricted arteries, and doesnot cause deleterious hypotension when administered to normotensivepatients.

FIG. 23 shows that relaxin mediated improvement in dyspnea is correlatedwith a normal or elevated baseline systolic blood pressure (SBP).

DETAILED DESCRIPTION General Overview

The present disclosure relates to methods of reducing decompensation inpopulations of subjects that are specifically prone to symptoms andevents of acute decompensated heart failure (AHF) such as dyspnea andfluid retention. Since AHF is the most common reason why patients over65 years of age are admitted to the hospital, it is associated withstaggering costs to the health care system. The prognosis for patientsthat are admitted with AHF or symptoms thereof has so far been dismal asit is associated with high readmission and mortality rates within sixmonths of admission. As disclosed herein, when patients who havepreviously been diagnosed with AHF and/or acute vascular failure orexhibit symptoms that are typical of AHF and/or acute vascular failureare treated with relaxin, their condition improves markedly andstabilizes over a short period of time. More specifically, when relaxinis administered to subjects who suffer from acute decompensationassociated with AHF, significant cardiovascular and renal improvementsare seen in these subjects. For example, when patients were administeredrelaxin for as little as 48 hours the improvements lasted over a periodof 14 days. The improvements included significant reductions in acutecardiac decompensation events including a noticeable reduction indyspnea (shortness of breath), a reduction in excessive body weight dueto fluid retention (e.g., patients lost on the average about 1 kg ofbody weight), shorter hospital stays (e.g., by as much as 2.5 days), adecreased likelihood of hospital re-admissions, a lower need for loopdiuretics, a lower need for intravenous nitroglycerin and a decreasedincidence of worsening heart failure. These changes significantlyimproved patient well-being and have strong future implications onpharmacoeconomics including reductions in cost of care.

Without wanting to be bound by theory, relaxin is contemplated to actthrough specific receptors that are found on smooth muscle cells thatmake up the vasculature (FIG. 3). As such, relaxin is a specific,moderate, systemic and renal vasodilator that improves heart and renalfunction via specific and balanced vasodilation. Since AHF is acardio-renal disease, relaxin benefits patients afflicted with AHFand/or acute vascular failure and/or symptoms thereof.

DEFINITIONS

The term “relaxin” refers to a peptide hormone which is well known inthe art (see FIG. 1). The term “relaxin”, as used herein, encompasseshuman relaxin, including intact full length human relaxin or a portionof the relaxin molecule that retains biological activity. The term“relaxin” encompasses human H1 preprorelaxin, prorelaxin, and relaxin;H2 preprorelaxin, prorelaxin, and relaxin; and H3 preprorelaxin,prorelaxin, and relaxin. The term “relaxin” further includesbiologically active (also referred to herein as “pharmaceuticallyactive”) relaxin from recombinant, synthetic or native sources as wellas relaxin variants, such as amino acid sequence variants. As such, theterm contemplates synthetic human relaxin and recombinant human relaxin,including synthetic H1, H2 and H3 human relaxin and recombinant H1, H2and H3 human relaxin. The term further encompasses active agents withrelaxin-like activity, such as relaxin agonists and/or relaxin analogsand portions thereof that retain biological activity, including allagents that competitively displace bound relaxin from a relaxin receptor(e.g., LGR7 receptor, LGR8 receptor, GPCR135, GPCR142, etc.). Thus, apharmaceutically effective relaxin agonist is any agent withrelaxin-like activity that is capable of binding to a relaxin receptorto elicit a relaxin-like response. In addition, the nucleic acidsequence of human relaxin as used herein must not be 100% identical tonucleic acid sequence of human relaxin (e.g., H1, H2 and/or H3) but maybe at least about 40%, 50%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the nucleic acid sequence of human relaxin. Relaxin, asused herein, can be made by any method known to those skilled in theart. Examples of such methods are illustrated, for example, in U.S. Pat.No. 5,759,807 as well as in Büllesbach et al. (1991) The Journal ofBiological Chemistry 266(17):10754-10761. Examples of relaxin moleculesand analogs are illustrated, for example, in U.S. Pat. No. 5,166,191.Naturally occurring biologically active relaxin may be derived fromhuman, murine (i.e., rat or mouse), porcine, or other mammalian sources.Also encompassed is relaxin modified to increase in vivo half life,e.g., PEGylated relaxin (i.e., relaxin conjugated to a polyethyleneglycol), modifications of amino acids in relaxin that are subject tocleavage by degrading enzymes, and the like. The term also encompassesrelaxin comprising A and B chains having N- and/or C-terminaltruncations. In general, in H2 relaxin, the A chain can be varied fromA(1-24) to A(10-24) and B chain from B(1-33) to B(10-22); and in H1relaxin, the A chain can be varied from A(1-24) to A(10-24) and B chainfrom B(1-32) to B(10-22). Also included within the scope of the term“relaxin” are other insertions, substitutions, or deletions of one ormore amino acid residues, glycosylation variants, unglycosylatedrelaxin, organic and inorganic salts, covalently modified derivatives ofrelaxin, preprorelaxin, and prorelaxin. Also encompassed in the term isa relaxin analog having an amino acid sequence which differs from awild-type (e.g., naturally-occurring) sequence, including, but notlimited to, relaxin analogs disclosed in U.S. Pat. No. 5,811,395.Possible modifications to relaxin amino acid residues include theacetylation, formylation or similar protection of free amino groups,including the N-terminal, amidation of C-terminal groups, or theformation of esters of hydroxyl or carboxylic groups, e.g., modificationof the tryptophan (Trp) residue at B2 by addition of a formyl group. Theformyl group is a typical example of a readily-removable protectinggroup. Other possible modifications include replacement of one or moreof the natural amino-acids in the B and/or A chains with a differentamino acid (including the D-form of a natural amino-acid), including,but not limited to, replacement of the Met moiety at B24 with norleucine(Nle), valine (Val), alanine (Ala), glycine (Gly), serine (Ser), orhomoserine (HomoSer). Other possible modifications include the deletionof a natural amino acid from the chain or the addition of one or moreextra amino acids to the chain. Additional modifications include aminoacid substitutions at the B/C and C/A junctions of prorelaxin, whichmodifications facilitate cleavage of the C chain from prorelaxin; andvariant relaxin comprising a non-naturally occurring C peptide, e.g., asdescribed in U.S. Pat. No. 5,759,807. Also encompassed by the term“relaxin” are fusion polypeptides comprising relaxin and a heterologouspolypeptide. A heterologous polypeptide (e.g., a non-relaxinpolypeptide) fusion partner may be C-terminal or N-terminal to therelaxin portion of the fusion protein. Heterologous polypeptides includeimmunologically detectable polypeptides (e.g., “epitope tags”);polypeptides capable of generating a detectable signal (e.g., greenfluorescent protein, enzymes such as alkaline phosphatase, and othersknown in the art); therapeutic polypeptides, including, but not limitedto, cytokines, chemokines, and growth factors. All such variations oralterations in the structure of the relaxin molecule resulting invariants are included within the scope of this disclosure so long as thefunctional (biological) activity of the relaxin is maintained.Preferably, any modification of relaxin amino acid sequence or structureis one that does not increase its immunogenicity in the individual beingtreated with the relaxin variant. Those variants of relaxin having thedescribed functional activity can be readily identified using in vitroand in vivo assays known in the art.

The term “heart failure” generally means that the heart is not workingas efficiently as it should. Heart failure occurs when the heart musclecannot keep up with the needs the body has for blood flow. It is asyndrome, i.e., a collection of findings which may arise from a numberof causes. Heart failure can be caused by weakening of the heart muscle(i.e., cardiomyopathy), leaving it unable to pump enough blood. Heartfailure is also termed congestive heart failure (CHF) because fluidstypically build up in the body, which is then said to be congested. Inaddition to heart failure caused from a weakened heart, there are alsoother varieties of heart failure. These are CHF due to the body havingneeds which are too high for even a normal heart to keep up with, forexample, in some cases of thyroid disease in which too much thyroidhormone is produced, in patients with anemia, or several otherconditions; and CHF due to neurohormonal imbalances that eventuallyleads to acute episodes of dyspnea or other acute events such ashypertension, high blood pressure, arrhythmia, reduced renal blood flow,renal insufficiency and in severe cases mortality, shifting the patientfrom compensated CHF to acute decompensated heart failure (AHF) and/oracute vascular failure.

The terms “acute cardiac decompensation” and “acute decompensation” areused interchangeably herein, and mean for the purpose of thespecification and claims, an inability of the heart muscle to compensatefor systemic and renal vasoconstriction due to neurohormonal imbalancesin the body. Acute cardiac decompensation is characterized by alteredcardiac function and fluid regulation, leading to the onset ofhemodynamic instability and physiologic changes (particularly congestionand edema), and heart failure symptoms (most commonly dyspnea). Thisform of functional decompensation could be misdiagnosed as being causedby a valvular or myocardial defect (i.e., a structural defect) althoughit is not usually associated with hypotension. However, “acute cardiacdecompensation” as used herein, is a functional decompensation that isoften associated with any one or more of certain decompensation events,including but not limited to, dyspnea, hypertension, high bloodpressure, arrhythmia, reduced renal blood flow, renal insufficiency andmortality. Patients presenting with “acute cardiac decompensation”, asused herein, typically have, but may not have previously been diagnosedwith chronic heart failure (CHF). Such patients may have a history ofheart disease or the complete absence thereof.

“Administering” refers to giving or applying to a subject apharmaceutical remedy or formulation via a specific route, including butnot limited to, intravenously, subcutaneously, intramuscularly,sublingually and via inhalation.

The term “vasculature” refers to the network of blood vessels in anorgan or body part, including arteries and capillaries.

The term “balanced vasodilation” means, for purpose of the specificationand claims, a dual vasodilation that occurs in the systemic (mostlyarterial) and renal vasculature as a result of the binding of relaxin ora relaxin agonist to specific relaxin receptors.

The terms “neurohormonal imbalance” and “neurohumoral imbalance” areused interchangeably herein, and refer to a hormonal disturbance in thebody that can lead to heart failure. For example, excessive signalingthrough Gs-coupled adrenergic or Gq-coupled angiotensin pathways cancause neurohormonal imbalances. In both cases, excessive neurohormonalsignaling can cause, as well as accelerate, functional decompensation(see Schrier et al., supra). In addition, excessive neurohormonalsignaling can cause, as well as accelerate, acute vascular failure.

The term “fluid overload”, as used herein, refers to a condition thatoccurs when the blood contains too much water. Fluid overload(hypervolemia) is commonly seen with heart failure that can cause fluidoverload by activation of the renin-angiotensin-aldosterone system. Thisfluid, primarily salt and water, builds up in various locations in thebody and leads to an increase in weight, swelling in the legs and arms(peripheral edema), and/or in the abdomen (ascites). Eventually, thefluid enters the air spaces in the lungs, reduces the amount of oxygenthat can enter the blood, and causes shortness of breath (dyspnea).Fluid can also collect in the lungs when lying down at night and canmake night time breathing and sleeping difficult (paroxysmal nocturnaldyspnea). Fluid overload is one of the most prominent features of AHFand/or acute vascular failure.

The term “cardiac arrhythmia” means a condition where the musclecontraction of the heart becomes irregular. An unusually fast rhythm(more than 100 beats per minute) is called tachycardia. An unusuallyslow rhythm (fewer than 60 beats per minute) is called bradycardia.

“Cardiac ischemia” occurs when blood flow to the heart muscle(myocardium) is obstructed by a partial or complete blockage of acoronary artery. A sudden, severe blockage may lead to a heart attack(myocardial infarction). Cardiac ischemia may also cause a seriousabnormal heart rhythm (arrhythmia), which can cause fainting and insevere cases death.

The term “pathophysiological” refers to a disturbance of any normalmechanical, physical, or biochemical function, either caused by adisease, or resulting from a disease or abnormal syndrome or conditionthat may not qualify to be called a disease. “Pathophysiology” is thestudy of the biological and physical manifestations of disease as theycorrelate with the underlying abnormalities and physiologicaldisturbances.

The term “nitric oxide” and “NO” are used interchangeably herein andrefer to an important signaling molecule involved in many physiologicaland pathological processes within the mammalian body, including inhumans. NO can act as a vasodilator that relaxes the smooth muscle inblood vessels, which causes them to dilate. Dilation of arterial bloodvessels (mainly arterioles) leads to a decrease in blood pressure.Relaxin is believed to elicit at least some vasodilation through NO. Assuch, relaxin binds to specific relaxin receptors such as LGR7 and LGR8receptors on smooth muscle cells of the vasculature which in turnactivates the endothelin cascade to activate nitric oxide synthase (NOS)to produce NO (FIG. 2).

The terms “AHF,” “acute heart failure” and “acute decompensated heartfailure” as used herein is defined by the presence of all of thefollowing at screening: dyspnea at rest or with minimal exertion,pulmonary congestion on chest X-ray and elevated natriuretic peptidelevels [brain natriuretic peptide (BNP)≧350 pg/mL or NT-pro-BNP≧1400pg/mL].

The term “dyspnea” refers to difficult or labored breathing. It is asign of a variety of disorders and is primarily an indication ofinadequate ventilation or of insufficient amounts of oxygen in thecirculating blood. The term “orthopnea” refers to difficult or laboredbreathing when lying flat, which is relieved when in an upright position(sitting or standing as opposed to reclining).

Clinical studies and practice guidelines typically define hypertensionas a systolic blood pressure (SBP) greater than about 140 mm Hg, andnormal blood pressure as a SBP below about 140 mm Hg, 130 mm Hg or 120mm Hg, depending upon the particular study or guideline. In the contextof acute heart failure or other cardiac disease, hypotension may becharacterized as a SBP below about 110 mm Hg, 100 mm Hg, or 90 mm Hg. Insome preferred embodiments, the phrase a “normotensive or hypertensivestate” refers to a SBP of greater than 125 mmHg at the time of studyscreening or relaxin administration.

As used herein, the phrase “impaired renal function” is defined as anestimated glomerular filtration rate (eGFR) of between 30 to 75mL/min/1.73 m2, calculated using the simplified Modification of Diet inRenal Disease (sMDRD) equation.

The term “placebo” refers to a physiologically inert treatment that isoften compared in clinical research trials to a physiologically activetreatment. These trials are usually carried out as double blind studiesand neither the prescribing doctor nor the patients know if they aretaking the active drug or the substance without any apparentpharmaceutical effect (placebo). It has been observed that a patientreceiving a physiologically inert treatment can demonstrate improvementfor his or her condition if he or she believes they are receiving thephysiologically active treatment (placebo effect). Therefore, theinclusion of a placebo in a trial assures that the statisticallysignificant beneficial effect is related to the physiologically activetreatment and not simply a result of a placebo effect.

The definition of “rehospitalization” is a hospital readmission during acertain time period after initial treatment. The time period isgenerally dependent on the kind of treatment and the condition of thepatient.

As used herein the term “cardiovascular death” refers to death that isprimarily due to a cardiovascular cause, such as death due to stroke,acute myocardial infarction, refractory congestive heart failure and anysudden.

A “loop diuretic” means a drug used in patients with congestive heartfailure or renal insufficiency to reduce symptoms of hypertension andedema. A loop diuretic belongs to a class of diuretic agents thatreduces readsorption of sodium and chloride by the kidney leading to anincreased secretion of urine.

The term “about” when used in the context of a stated value, encompassesa range of up to 10% above or below the stated value (e.g., 90-110% ofthe stated value). For instance, an intravenous (IV) infusion rate ofabout 30 mcg/kg/day, encompasses IV infusion rates of 27 mcg/kg/day to33 mcg/kg/day.

“Therapeutically effective” refers to the amount of pharmaceuticallyactive relaxin that will result in a measurable desired medical orclinical benefit to a patient, as compared to the patient's baselinestatus or to the status of an untreated or placebo-treated (e.g., nottreated with relaxin) subject.

Relaxin

Relaxin is a polypeptide hormone that is similar in size and shape toinsulin (FIG. 1). More specifically, relaxin is an endocrine andautocrine/paracrine hormone belonging to the insulin gene superfamily.The active form of the encoded protein consists of an A chain and a Bchain, held together by disulphide bonds, two inter-chains and oneintra-chain. Thus, the structure closely resembles insulin in thedisposition of disulphide bonds. In humans, there are three knownnon-allelic relaxin genes, relaxin-1 (RLN-1 or H1), relaxin-2 (RLN-2 orH2) and relaxin-3 (RLN-3 or H3). H1 and H2 share high sequence homology.There are two alternatively spliced transcript variants encodingdifferent isoforms described for this gene. H1 and H2 are differentiallyexpressed in reproductive organs (U.S. Pat. No. 5,023,321 andGaribay-Tupas et al., Molecular and Cellular Endocrinology 219:115-125,2004), while H3 is found primarily in the brain. The evolution of therelaxin peptide family in its receptors is generally well known in theart (Wilkinson et al., BMC Evolutionary Biology 5(14):1-17, 2005; andWilkinson and Bathgate, Chapter 1, Relaxin and Related Peptides, LandesBioscience and Springer Science+Business Media, 2007).

Relaxin activates specific relaxin receptors, i.e., LGR7 (RXFP1) andLGR8 (RXFP2) as well as GPCR135 and GPCR142. LGR7 and LGR8 areleucine-rich repeat-containing, G protein-coupled receptors (LGRs) whichrepresent a unique subgroup of G protein-coupled receptors. They containa heptahelical transmembrane domain and a large glycosylated ectodomain,distantly related to the receptors for the glycoproteohormones, such asthe LH-receptor or FSH-receptor. These relaxin receptors are found inthe heart, smooth muscle, connective tissue, and central and autonomousnervous system. Potent relaxins such as H1, H2, porcine and whalerelaxin possess a certain sequence in common, i.e., theArg-Glu-Leu-Val-Arg-X-X-Ile sequence (SEQ ID NO:3) or binding cassette.These relaxins activate the LGR7 and LGR8 receptors. Relaxins thatdeviate from this sequence homology such as rat, shark, dog and horserelaxins show a reduction in bioactivity through the LGR7 and LGR8receptors (see Bathgate et al. (2005) Ann. N.Y. Acad. Sci. 1041:61-76;Receptors for Relaxin Family Peptides). However, similar to H2 relaxin,H3 relaxin activates the LGR7 receptor (see Satoko et al. (2003) TheJournal of Biological Chemistry 278(10):7855-7862). In addition, H3 hasbeen shown to activate the GPCR135 receptor (see Van der Westhuizen(2005) Ann. N.Y. Acad. Sci. 1041:332-337) and GPCR142 receptor. GPCR135and GPCR142 are two structurally related G-protein-coupled receptors.Mouse and rat GPCR135 exhibit high homology (i.e., greater than 85%) tothe human GPCR135 and have very similar pharmacological properties tothat of the human GPCR135. Human and mouse as well as rat relaxin-3binds to and activates mouse, rat, and human GPCR135 at high affinity.In contrast, the mouse GPCR142 is less well conserved (i.e., 74%homology) with human GPCR142. GPCR142 genes from monkey, cow, and pigwere cloned and shown to be highly homologous (i.e., greater than 84%)to human GPCR142. Pharmacological characterization of GPCR142 fromdifferent species has shown that relaxin-3 binds to GPCR142 fromdifferent species at high affinity (see Chen et al. (2005) The Journalof Pharmacology and Experimental Therapeutics 312(1):83-95).

Relaxin is found in both, women and men (see Tregear et al.; Relaxin2000, Proceedings of the Third International Conference on Relaxin &Related Peptides (22-27 Oct. 2000, Broome, Australia). In women, relaxinis produced by the corpus luteum of the ovary, the breast and, duringpregnancy, also by the placenta, chorion, and decidua. In men, relaxinis produced in the testes. Relaxin levels rise after ovulation as aresult of its production by the corpus luteum and its peak is reachedduring the first trimester, not toward the end of pregnancy. In theabsence of pregnancy its level declines. In humans, relaxin is plays arole in pregnancy, in enhancing sperm motility, regulating bloodpressure, controlling heart rate and releasing oxytocin and vasopressin.In animals, relaxin widens the pubic bone, facilitates labor, softensthe cervix (cervical ripening), and relaxes the uterine musculature. Inanimals, relaxin also affects collagen metabolism, inhibiting collagensynthesis and enhancing its breakdown by increasing matrixmetalloproteinases. It also enhances angiogenesis and is a renalvasodilator.

Relaxin has the general properties of a growth factor and is capable ofaltering the nature of connective tissue and influencing smooth musclecontraction. H1 and H2 are believed to be primarily expressed inreproductive tissue while H3 is known to be primarily expressed in brain(supra). As disclosed herein, H2 plays a major role in cardiovascularand cardiorenal function and can thus be used to treat associateddiseases. H1 and H3 due to their homology with H2 are contemplated to besuitable for treating cardiovascular disease. In addition,pharmaceutically effective relaxin agonists with relaxin-like activitywould be capable of activating relaxin receptors and to elicit arelaxin-like response.

Acute Heart Failure (AHF) Patients

AHF is the most common cause for hospital admission in patients olderthan 65 years and for congestive heart failure-related morbidity (Cotteret al., American Heart Journal 155(1):9-18, 2008). In spite of theprogress made in mortality-reducing drug therapies for chronic(systolic) heart failure, including angiotensin-converting enzymeinhibitors, angiotensin II receptor blockers, β-blockers, andaldosterone antagonists, no comparable progress has been made in the artfor AHF, where both therapy and mortality have not changed significantlyover the past 30 years (Allen et al., CMAJ 176:797-805, 2007). Theclassic AHF drugs such as loop diuretics, nitroglycerin/nitroprusside,dobutamine, or milrinone have not been able to improve AHF outcome(Allen et al., supra). The same is true for therapeutic strategiesincluding endothelin-1 receptor blockade with TEZOSENTAN, vasopressin V2receptor antagonism using TOLVAPTAN, the natriuretic peptide NESIRITIDE,and LEVOSIMENDAN which combines calcium-sensitizing and vasodilatoryproperties. Chronic renal dysfunction is frequently a part of thecomplex morbidity of AHF, particularly in older AHF patients.Deterioration of renal function can induce or worsen AHF (i.e.,cardiorenal syndrome) and is related to significant morbidity in the AHFpopulation. According to the ADHERE registry (Heywood, Heart Fail. Rev.9:195-201, 2004), impairment of renal function correlates with a worseprognosis for AHF. Hence, treatment with relaxin provides a novel AHFtherapy with favorable renal effects, which significantly improves theprognosis for patients that are part of the AHF population. Inaccordance, pharmaceutically active relaxin can be used to treat theseAHF patients, or subjects afflicted with acute cardiac decompensationevents or symptoms, or subjects afflicted with acute cardiacdecompensation that is associated with AHF.

Patients with AHF can be classified into three groups based on theirsystolic blood pressure at the time of presentation (See, e.g.,Gheorghiade et al., JAMA, 296: 2217-2226, 2006; and Shin et al., Am JCardiol, 99[suppl]:4A-23A. 2007). The three groups include: 1) thehypotensive group (low blood pressure); 2) the normotensive group(normal blood pressure) and 3) the hypertensive group (high bloodpressure).

Hypotensive AHF patients having a very low left ventricle ejectionfraction (LVEF) are described as having “low cardiac output” or“cardiogenic shock.” Such hypotensive AHF patients have hearts that failto adequately pump blood, meaning that the percentage of the blood inthe ventricle that is pumped out with each contraction is reduced.

Normotensive AHF patients have higher blood pressure and typically agreater LVEF than hypotensive AHF patients and are sometimes describedas having “cardiac failure.” The cause of AHF in these patients is acombination of both depressed cardiac function and vasoconstriction.

Hypertensive AHF patients have higher blood pressure and typically agreater LVEF than normotensive AHF patients and are generally describedas having “vascular failure.” Even though these patients have somedegree of abnormal cardiac function, the predominant cause of their AHFis vasoconstriction.

Current data indicates that vascular failure and cardiac failure may bethe most common types of AHF, as opposed to low cardiac output (ADHEREScientific Advisory Committee, Acute Decompensated Heart FailureNational Registry (ADHERE) Core Module Q1 2006 Final Cumulative NationalBenchmark Report, Scios, Inc. pp. 1-19, 2006). Many patients presentingwith acute heart failure signs and symptoms, including pulmonarycongestion on x-ray, difficulty breathing (dyspnea) and normal(normotensive) or high (hypertensive) blood pressure have preserved leftventricular function (generally >40% EF). These acute heart failurepatients exhibit problems with excessive vasoconstriction and withfilling the ventricle with blood, rather than the ability of theventricle to pump blood. These patients are clearly distinguishable fromhypotensive AHF patients.

Traditional treatment of low cardiac output or cardiogenic shock(hypotensive AHF) involve pharmacologic agents that cause the heart tocontract harder (inotropic) and/or faster (chronotropic) to maintain theperfusion of vital organs (See, e.g., Nieminen et al, Eur Heart J,26:384-416, 2005; and Shin et al, Am J Cardiol, 99[suppl]:4A-23A. 2007).However, normotensive (vascular failure) and hypertensive (cardiacfailure) HF patients are extremely sensitive to changes in heart ratesince an increase in heart rate reduces the filling time betweenvascular contractions, and hence lowers the volume of blood filling theventricle (Satpathy et al., American Family Physician, 73:841-846,2006). For this reason, treatments for acute heart failure that increaseheart rate would be detrimental to hypertensive acute heart failurepatients, if not contraindicated (Satpathy et al., supra)

Generally, pharmaceutically effective relaxin or a pharmaceuticallyeffective relaxin agonist should be administered at a constant rate toprovide safe relief and achieve a steady state in the patient. Forexample, it is preferred to administer relaxin intravenously to maintaina serum concentration of relaxin of from about 1 to 500 ng/ml. Morespecifically, the administration of relaxin is continued as to maintaina serum concentration of relaxin of from about 0.5 to about 500 ng/ml,more preferably from about 0.5 to about 300 ng/ml, and most preferablyfrom about 3 to about 75 ng/ml. The subject would be treated withrelaxin at about 10 to 1000 μg/kg of subject body weight per day ratherthan via a loading dose such as a bolus. More preferably, the subjectwould be treated with relaxin at about 10 to about 250 μg/kg of subjectbody weight per day. In another preferred embodiment, pharmaceuticallyeffective relaxin or an agonist thereof is administered at about 30μg/kg/day. Other forms of administering relaxin are also contemplated bythe disclosure including, but not limited to, subcutaneously,intramuscularly, sublingually and via inhalation. Notably, acutesituations are normally treated with a loading dose (bolus) because thepatient is “acute” and needs instant relief. However, this can lead tosituation where the patient is overcompensated, thus, leading toworsening of heart failure symptoms or even death. As described herein,administering relaxin at a constant rate in acute situations is a safeand effective form of administration.

Relaxin Treatment Results in Balanced Vasodilation

Without wanting to be bound by theory, the beneficial effect of relaxinis believed to be a direct result of relaxin acting as areceptor-specific vasodilator in the renal and systemic vasculature bybinding to specific relaxin receptors that are found on the smoothmuscle tissue of the vasculature. This in turn results in balancedvasodilation as both systemic and renal arteries are vasodilated in amoderate but effective way without causing hypotension in the treatedpatient. This property of relaxin as a receptor-specific and balancingvasodilator is particularly advantageous in context in which it isdesirable to obtain increased vasodilation in specific areas of the bodywhere vasoconstriction causes a serious ill effect such as in thearteries that supply blood to the heart and the kidneys. Notably, thebalanced vasodilation occurs without causing any deleterious side effectduring the process of treatment. A common problem with treatment withnon-specific vasodilators is that these drugs often lead to serious sideeffects in the treated patients, mainly because general agonists act toopotently and non-specifically. In comparison, the moderate effect ofrelaxin slowly increases vasodilation in areas of the body where it isneeded the most. It is important to note that relaxin treatment does notcause hypotension as is the case with many drugs that overcompensate forvasoconstriction. In particular, non-specific vasodilators can causelarge and small arteries and veins throughout the body to dilateexcessively, causing hypotension. Thus, when the patient receives apharmaceutical composition with pharmaceutically active relaxin orpharmaceutically effective relaxin agonist which targets systemic andrenal blood vessels via localized specific relaxin receptors (e.g.,LRG7, LGR8, GPCR135, GPCR142 receptors) the result is balancedvasodilation without hypotension.

Consequently, relaxin can be used to reduce cardiac decompensationevents by selecting human subjects including AHF patients and/orindividuals with AHF symptoms and/or individuals suffering from acutevascular failure who present with acute cardiac decompensation, andadministering to those subjects a pharmaceutical formulation withpharmaceutically active relaxin. Relaxin reduces the acute cardiacdecompensation events by binding to the relaxin receptors (e.g., LRG7,LGR8, GPCR135, GPCR142 receptors) resulting in balanced vasodilation,i.e., a dual vasodilation in both the systemic and renal vasculature.Based on those same principles, relaxin can be used to treat cardiacdecompensation in human subjects including AHF patients and/orindividuals associated with symptoms of AHF and/or individuals sufferingfrom acute vascular failure. Particularly, such subjects receivepharmaceutically active human relaxin (e.g., synthetic, recombinant) orpharmaceutically effective relaxin agonist in an amount in a range ofabout 10 to 1000 μg/kg of subject body weight per day. In oneembodiment, the dosages of relaxin are 10, 30, 100 and 250 μg/kg/day. Inanother embodiment, these dosages result in serum concentrations ofrelaxin of about 3, 10, 30 and 75 ng/ml, respectively. In one preferredembodiment, pharmaceutically effective relaxin or an agonist thereof isadministered at about 30 μg/kg/day. In another preferred embodiment,pharmaceutically effective relaxin or an agonist thereof is administeredat about 10 to about 250 μg/kg/day. In another embodiment, theadministration of relaxin is continued as to maintain a serumconcentration of relaxin of from about 0.5 to about 500 ng/ml, morepreferably from about 0.5 to about 300 ng/ml, and most preferably fromabout 3 to about 75 ng/ml. Most preferably, the administration ofrelaxin is continued as to maintain a serum concentration of relaxin ofabout 10 ng/ml or greater. Relaxin has also been shown to be fullyeffective at a serum concentration of 3-6 ng/ml. Thus, the methods ofthe present disclosure include administrations that result in theseserum concentrations of relaxin. These relaxin concentrations canameliorate or reduce decompensation events such as dyspnea,hypertension, arrhythmia, reduced renal blood flow, and renalinsufficiency. Furthermore, these relaxin concentrations can ameliorateor reduce neurohormonal imbalance, fluid overload, cardiac arrhythmia,cardiac ischemia, risk of mortality, cardiac stress, vascularresistance, and the like.

The duration of relaxin treatment is preferably kept at a range of about4 hours to about 96 hours depending on the patient, and one or moreoptional repeat treatments as needed. For example, with respect tofrequency of administration, relaxin administration can be a continuousinfusion lasting from about 8 hr to 72 hours of treatment. Relaxin canbe given continuously via intravenous or subcutaneous administration.For intravenous administration, relaxin can be delivered by syringe pumpor through an IV bag. The IV bag can be a standard saline, half normalsaline, 5% dextrose in water, lactated Ringer's or similar solution in a100, 250, 500 or 1000 ml IV bag. For subcutaneous infusion, relaxin canbe administered by a subcutaneous infusion set connected to a wearableinfusion pump. Depending on the subject, the relaxin administration ismaintained for as specific period of time or for as long as needed toachieve stability in the subject.

Some subjects are treated indefinitely while others are treated forspecific periods of time. It is also possible to treat a subject on andoff with relaxin as needed. Thus, administration can be continued over aperiod of time sufficient to achieve an amelioration or reduction inacute cardiac decompensation events, including but not limited to,dyspnea, hypertension, arrhythmia, reduced renal blood flow and renalinsufficiency. Relaxin may be administered in higher doses if necessaryto prevent death due to AHF and/or acute vascular failure associatedcomplications such as sudden cardiac arrest.

Relaxin Treatment does not Cause Renal Toxicity and is Diuretic-Sparing

Renal dysfunction is a common and progressive complication of acute andchronic heart failure. The clinical course typically fluctuates with thepatient's clinical status and treatment. Despite the growing recognitionof the frequent presentation of combined cardiac and renal dysfunction,also termed the “cardiorenal syndrome,” its underlying pathophysiologyis not well understood. No consensus as to its appropriate managementhas been achieved in the art. Because patients with heart failure aresurviving longer and die less frequently from cardiac arrhythmia,cardiorenal syndrome is more and more prevalent and proper management isneeded (Gary Francis (2006) Cleveland Clinic Journal of Medicine73(2):1-13). The disclosure solves this need. It provides a method oftreating acute decompensated heart failure (AHF) and/or acute vascularfailure in a human subject who also suffers from renal insufficiency.This method includes selecting a human subject with symptoms of acutecardiac decompensation and renal insufficiency, wherein the subject hasa systemic and renal vasculature comprising relaxin receptors. Relaxinis administered to the subject and performs a dual action by binding tothe relaxin receptors in the systemic and renal vasculature, resultingin balanced vasodilation. As noted above, such subjects receivepharmaceutically active human relaxin (e.g., synthetic, recombinant) orpharmaceutically effective relaxin agonist in an amount in a range ofabout 10 to 1000 μg/kg of subject body weight per day. In oneembodiment, the dosages of relaxin are 10, 30, 100 and 250 μg/kg/day. Inanother embodiment, these dosages result in serum concentrations ofrelaxin of about 3, 10, 30 and 75 ng/ml, respectively. In one preferredembodiment, pharmaceutically effective relaxin or an agonist thereof isadministered at about 30 μg/kg/day. In another preferred embodiment,pharmaceutically effective relaxin or an agonist thereof is administeredat about 10 to about 250 μg/kg/day. The administration of relaxin iscontinued as to maintain a serum concentration of relaxin of from about0.5 to about 500 ng/ml, more preferably from about 0.5 to about 300ng/ml, and most preferably from about 3 to about 75 ng/ml. Mostpreferably, the administration of relaxin is continued as to maintain aserum concentration of relaxin of 10 ng/ml or greater. Depending on thesubject, the relaxin administration is maintained for as specific periodof time or for as long as needed to achieve stability in the subject.For example, the duration of relaxin treatment is preferably kept at arange of about 4 hours to about 96 hours, more preferably from about 8hr to 72 hours, depending on the patient, and one or more optionalrepeat treatments as needed.

Subjects who suffer from renal insufficiency associated with AHF oftenalso experience elevated levels of brain natriuretic peptide (BNP). BNPis synthesized in the cardiac ventricles in response to heart failureand left ventricular dysfunction. It is used as a diagnostic marker ofheart failure. Its effects include systemic vasodilation and unbalancedvasodilation in the kidney, i.e., efferent arteriolar constriction andafferent arteriole vasodilation. As described herein, brain natriureticpeptide (BNP) levels are reduced when relaxin is administered to AHFpatients and/or patients with acute vascular failure. This makes BNP aconvenient AHF marker since it is reduced as the severity of AHF isreduced. Monitoring BNP levels in patients that are treated with relaxinis, thus, a convenient way to assess the risk of mortality associatedwith AHF and/or acute vascular failure. Thus, the disclosure provides amethod for reducing mortality risk in a human subject with symptoms ofacute cardiac decompensation. The relaxin is administered in an amounteffective to reduce the acute cardiac decompensation in the subject bybinding to the relaxin receptors in the vasculature of the subject,thereby resulting in reduced levels of BNP. The reduced levels of BNPcan be physically measured in order to predict risk of mortality in theAHF and/or acute vascular failure patient. Generally, the reduced levelsof BNP are due to reduced cardiac stress following a reduction invascular resistance. The reduction in vascular resistance is in turn dueto the balanced vasodilation which is the result of relaxin binding torelaxin receptors that are found on smooth muscle cells of thevasculature.

Relaxin causes low to no renal toxicity when it is given to AHF and/oracute vascular failure patients in comparison to most available drugs.Even with higher serum concentrations of about 75 ng/ml relaxin is farless toxic than currently available medications (e.g., loop diureticssuch as furosemide, angiotensin converting enzyme inhibitors such ascaptopril, angiotensin receptor blockers such as candesartan, and thelike). One important feature of this disclosure is that relaxinpreserves the renal function while causing little to no renal toxicityduring treatment. Although existing drugs may preserve some renalfunction they also increase renal toxicity in patients. This renaltoxicity then further deteriorates the heart condition. In comparison,relaxin will achieve a steady-state maintenance of most patients due tothe absence of renal toxicity. This allows the unstable AHF and/or acutevascular failure population to revert back to a more stable CHFpopulation or to achieve a stable condition where the likelihood ofexacerbating heart failure is significantly reduced.

Relaxin Compositions and Formulations

Relaxin, relaxin agonists and/or relaxin analogs are formulated aspharmaceuticals to be used in the methods of the disclosure. Anycomposition or compound that can stimulate a biological responseassociated with the binding of biologically or pharmaceutically activerelaxin (e.g., synthetic relaxin, recombinant relaxin) or a relaxinagonist (e.g., relaxin analog or relaxin-like modulator) to relaxinreceptors can be used as a pharmaceutical in the disclosure. Generaldetails on techniques for formulation and administration are welldescribed in the scientific literature (see Remington's PharmaceuticalSciences, Maack Publishing Co, Easton Pa.). Pharmaceutical formulationscontaining pharmaceutically active relaxin can be prepared according toany method known in the art for the manufacture of pharmaceuticals. Theformulations containing pharmaceutically active relaxin or relaxinagonists used in the methods of the disclosure can be formulated foradministration in any conventionally acceptable way including, but notlimited to, intravenously, subcutaneously, intramuscularly,sublingually, topically, orally and via inhalation. Illustrativeexamples are set forth below. In one preferred embodiment, relaxin isadministered intravenously.

When the drugs are delivered by intravenous injection, the formulationscontaining pharmaceutically active relaxin or a pharmaceuticallyeffective relaxin agonist can be in the form of a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated according to the known artusing those suitable dispersing or wetting agents and suspending agentswhich have been mentioned above. The sterile injectable preparation canalso be a sterile injectable solution or suspension in a nontoxicparenterally-acceptable diluent or solvent. Among the acceptablevehicles and solvents that can be employed are water and Ringer'ssolution, an isotonic sodium chloride. In addition, sterile fixed oilscan conventionally be employed as a solvent or suspending medium. Forthis purpose any bland fixed oil can be employed including syntheticmono- or diglycerides. In addition, fatty acids such as oleic acid canlikewise be used in the preparation of injectables.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical formulations to be formulated in unit dosage forms astablets, pills, powder, capsules, liquids, lozenges, gels, syrups,slurries, suspensions, etc., suitable for ingestion by the patient.Pharmaceutical preparations for oral use can be obtained throughcombination of relaxin compounds with a solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable additional compounds, if desired, to obtaintablets or pills. Suitable solid excipients are carbohydrate or proteinfillers which include, but are not limited to, sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; as well as proteins such asgelatin and collagen. If desired, disintegrating or solubilizing agentsmay be added, such as the cross-linked polyvinyl pyrrolidone, agar,alginic acid, or a salt thereof, such as sodium alginate. Pharmaceuticalpreparations of the disclosure that can also be used orally are, forexample, push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a coating such as glycerol or sorbitol.Push-fit capsules can contain relaxin mixed with a filler or binderssuch as lactose or starches, lubricants such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the relaxincompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

Aqueous suspensions of the disclosure contain relaxin in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Oil suspensions can be formulated by suspending relaxin in a vegetableoil, such as arachis oil, olive oil, sesame oil or coconut oil, or in amineral oil such as liquid paraffin. The oil suspensions can contain athickening agent, such as beeswax, hard paraffin or cetyl alcohol.Sweetening agents can be added to provide a palatable oral preparation.These formulations can be preserved by the addition of an antioxidantsuch as ascorbic acid.

Dispersible powders and granules of the disclosure suitable forpreparation of an aqueous suspension by the addition of water can beformulated from relaxin in admixture with a dispersing, suspendingand/or wetting agent, and one or more preservatives. Suitable dispersingor wetting agents and suspending agents are exemplified by thosedisclosed above. Additional excipients, for example sweetening,flavoring and coloring agents, can also be present.

The pharmaceutical formulations of the disclosure can also be in theform of oil-in-water emulsions. The oily phase can be a vegetable oil,such as olive oil or arachis oil, a mineral oil, such as liquidparaffin, or a mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening and flavoring agents. Syrups andelixirs can be formulated with sweetening agents, such as glycerol,sorbitol or sucrose. Such formulations can also contain a demulcent, apreservative, a flavoring or a coloring agent.

Administration and Dosing Regimen of Relaxin Formulations

The formulations containing pharmaceutically active relaxin orpharmaceutically effective relaxin agonist used in the methods of thedisclosure can be administered in any conventionally acceptable wayincluding, but not limited to, intravenously, subcutaneously,intramuscularly, sublingually, topically, orally and via inhalation.Administration will vary with the pharmacokinetics and other propertiesof the drugs and the patients' condition of health. General guidelinesare presented below.

The methods of the disclosure reduce acute cardiac decompensation eventsin subjects who suffer from acute cardiac decompensation associated withAHF and/or acute vascular failure, and/or related conditions. Inaddition, the methods of the disclosure treat acute cardiacdecompensation in subjects who suffer from acute cardiac decompensationassociated with AHF, including AHF patients and/or patients with acutevascular failure. The amount of relaxin alone or in combination withanother agent or drug (e.g., diuretic) that is adequate to accomplishthis is considered the therapeutically effective dose. The dosageschedule and amounts effective for this use, i.e., the “dosing regimen,”will depend upon a variety of factors, including the stage of thedisease or condition, the severity of the disease or condition, theseverity of the adverse side effects, the general state of the patient'shealth, the patient's physical status, age and the like. In calculatingthe dosage regimen for a patient, the mode of administration is alsotaken into consideration. The dosage regimen must also take intoconsideration the pharmacokinetics, i.e., the rate of absorption,bioavailability, metabolism, clearance, and the like. Based on thoseprinciples, relaxin can be used to treat cardiac decompensation in humansubjects including AHF patients and/or individuals associated withsymptoms of AHF and/or individuals who suffer from acute vascularfailure.

The disclosure provides relaxin and a diuretic for simultaneous,separate or sequential administration. The disclosure also providesrelaxin and a diuretic for combined use in therapy. The disclosure alsoprovides the combination of relaxin and a diuretic for use in therapy.The disclosure also provides the use of relaxin and a diuretic in themanufacture of a medicament for treating acute cardiac decompensationevents. The disclosure also provides the use of relaxin in themanufacture of a medicament for treating acute cardiac decompensationevents, wherein the medicament is prepared for administration with adiuretic. The disclosure also provides the use of a diuretic in themanufacture of a medicament for treating acute cardiac decompensationevents, wherein the medicament is prepared for administration withrelaxin. The disclosure also provides relaxin and a diuretic for use ina method of treating acute cardiac decompensation events.

The disclosure further provides relaxin for use in a method of treatingacute cardiac decompensation events, wherein relaxin is prepared foradministration with a diuretic. The disclosure also provides a diureticfor use in a method of treating acute cardiac decompensation events,wherein relaxin is prepared for administration with relaxin. Thedisclosure also provides relaxin for use in a method of treating acutecardiac decompensation events, wherein relaxin is administered with adiuretic. The disclosure also provides a diuretic for use in a method oftreating acute cardiac decompensation events, wherein relaxin isadministered with relaxin.

Further contemplates is the use of relaxin in the manufacture of amedicament for treating acute cardiac decompensation events, wherein thepatient has previously (e.g., a few hours before, one or more daysbefore, etc.) been treated with a diuretic. In one embodiment, thediuretic is still active in vivo in the patient. The disclosure alsoprovides the use of a diuretic in the manufacture of a medicament fortreating acute cardiac decompensation events, wherein the patient haspreviously been treated with relaxin.

The state of the art allows the clinician to determine the dosageregimen of relaxin for each individual patient. As an illustrativeexample, the guidelines provided below for relaxin can be used asguidance to determine the dosage regimen, i.e., dose schedule and dosagelevels, of formulations containing pharmaceutically active relaxinadministered when practicing the methods of the disclosure. As a generalguideline, it is expected that the daily dose of pharmaceutically activeH1, H2 and/or H3 human relaxin (e.g., synthetic, recombinant, analog,agonist, etc.) is typically in an amount in a range of about 10 to 1000μg/kg of subject body weight per day. In one embodiment, the dosages ofrelaxin are 10, 30, 100 and 250 μg/kg/day. In another embodiment, thesedosages result in serum concentrations of relaxin of about 3, 10, 30 and75 ng/mL, respectively. In one preferred embodiment, pharmaceuticallyeffective relaxin or an agonist thereof is administered at about 30μg/kg/day. In another preferred embodiment, pharmaceutically effectiverelaxin or an agonist thereof is administered at about 10 to about 250μg/kg/day. In another embodiment, the administration of relaxin iscontinued as to maintain a serum concentration of relaxin of from about0.5 to about 500 ng/ml, more preferably from about 0.5 to about 300ng/ml, and most preferably from about 3 to about 75 ng/ml. Mostpreferably, the administration of relaxin is continued as to maintain aserum concentration of relaxin of 10 ng/ml or greater. Relaxin has alsobeen shown to be fully effective at a serum concentration of 3-6 ng/ml(see FIG. 6, vide infra). Thus, the methods of the present disclosureinclude administrations that result in these serum concentrations ofrelaxin. These relaxin concentrations can ameliorate or reducedecompensation events such as dyspnea, hypertension, high bloodpressure, arrhythmia, reduced renal blood flow, renal insufficiency andmortality. Furthermore, these relaxin concentrations can ameliorate orreduce neurohormonal imbalance, fluid overload, cardiac arrhythmia,cardiac ischemia, risk of mortality, cardiac stress, vascularresistance, and the like. Depending on the subject, the relaxinadministration is maintained for as specific period of time or for aslong as needed to achieve stability in the subject. For example, theduration of relaxin treatment is preferably kept at a range of about 4hours to about 96 hours, more preferably 8 hours to about 72 hours,depending on the patient, and one or more optional repeat treatments asneeded.

Single or multiple administrations of relaxin formulations may beadministered depending on the dosage and frequency as required andtolerated by the patient who suffers from acute cardiac decompensation,AHF and/or conditions related to AHF and/or individuals suffering fromacute vascular failure. The formulations should provide a sufficientquantity of relaxin to effectively ameliorate the condition. A typicalpharmaceutical formulation for intravenous administration of relaxinwould depend on the specific therapy. For example, relaxin may beadministered to a patient through monotherapy (i.e., with no otherconcomitant medications) or in combination therapy with anothermedication such as a diuretic or other drug. In one embodiment, relaxinis administered to a patient daily as monotherapy. In anotherembodiment, relaxin is administered to a patient daily as combinationtherapy with another drug. Notably, the dosages and frequencies ofrelaxin administered to a patient may vary depending on age, degree ofillness, drug tolerance, and concomitant medications and conditions.

In some embodiments, relaxin is provided as a 1 mg/mL solution (3.5 mLin 5 mL glass vials). Placebo, which is identical to the diluent forrelaxin, is provided in identical vials. Relaxin or placebo isadministered intravenously to the patient in small volumes using asyringe pump in combination with normal saline in a piggybackconfiguration. Compatible tubing and a 3-way stopcock, which have beentested and qualified for use with relaxin are used to administer therelaxin formulation. Doses are administered on a weight basis andadjusted for each patient by adjusting the rate of relaxin drugdelivered by the infusion pump. In some embodiments, each subject isdosed for up to 48 hours with study drug.

Adjunct Therapies for Treating Normotensive and Hypertensive AHFPatients

There are a wide variety of approved antihypertensive drugs includingvasodilators, adrenergic blockers, centrally acting alpha-agonists,angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptorblockers (ARBs), calcium channel blockers and multiple types ofdiuretics (e.g., loop, potassium-sparing, thiazide and thiazide-like).In some embodiments, the present disclosure provides methods of treatingdyspnea associated with acute heart failure in normotensive andhypertensive patients comprising administration of relaxin incombination with an adjunct therapy such as an antihypertensive drug. Insome methods, the antihypertensive drug is selected from but not limitedto the following ACE inhibitors, beta-blockers and diuretics.

Angiotensin Converting Enzyme (ACE) inhibitors have been used for thetreatment of hypertension for many years. ACE inhibitors block theformation of angiotensin II, a hormone with adverse effects on the heartand circulation in CHF patients. Side effects of these drugs include adry cough, low blood pressure, worsening kidney function and electrolyteimbalances, and sometimes, allergic reactions. Examples of ACEinhibitors include captopril (CAPOTEN), enalapril (VASOTEC), lisinopril(ZESTRIL, PRINIVIL), benazepril (LOTENSIN), and ramipril (ALTACE). Forthose patients who are unable to tolerate ACE inhibitors, an alternativegroup of drugs, called the angiotensin receptor blockers (ARBs), can beused. These drugs act on the same hormonal pathway as ACE inhibitors,but instead block the action of angiotensin II at its receptor sitedirectly. Side effects of these drugs are similar to those associatedwith ACE inhibitors, although the dry cough is less common. Examples ofthis class of medications include losartan (COZAAR), candesartan(ATACAND), telmisartan (MICARDIS), valsartan (DIOVAN), and irbesartan(AVAPRO).

Beta-blockers are drugs that block the action of certain stimulatinghormones, such as epinephrine (adrenaline), norepinephrine, and othersimilar hormones, which act on the beta receptors of various bodytissues. The natural effect of these hormones on the beta receptors ofthe heart is a more forceful contraction of the heart muscle.Beta-blockers are agents that block the action of these stimulatinghormones on the beta receptors. The stimulating effect of thesehormones, while initially useful in maintaining heart function, appearsto have detrimental effects on the heart muscle over time. Generally, ifCHF patients receive beta-blockers they are given at a very low dose atfirst which is then gradually increased. Side effects include fluidretention, low blood pressure, low pulse, and general fatigue andlightheadedness. Beta-blockers should also not be used in people withdiseases of the airways (e.g., asthma, emphysema) or very low restingheart rates. Carvedilol (COREG) has been the most thoroughly studieddrug in the setting of congestive heart failure and remains the onlybeta-blocker with FDA approval for the treatment of congestive heartfailure. However, research comparing carvedilol directly with otherbeta-blockers in the treatment of congestive heart failure is ongoing.Long acting metopropol (TOPROL XL) is also effective in patients withcongestive heart failure. Digoxin (LANOXIN) is naturally produced by theFoxglove flowering plant and has been used for treatment of CHF patientsfor a decade. Digoxin stimulates the heart muscle to contract moreforcefully. Side effects include nausea, vomiting, heart rhythmdisturbances, kidney dysfunction, and electrolyte abnormalities. Inpatients with significant kidney impairment the dose of digoxin needs tobe carefully adjusted and monitored.

Diuretics are often used in the treatment of CHF patients to prevent oralleviate the symptoms of fluid retention. These drugs help keep fluidfrom building up in the lungs and other tissues by promoting the flow offluid through the kidneys. Although they are effective in relievingsymptoms such as shortness of breath and leg swelling, they have notbeen demonstrated to positively impact long term survival. Whenhospitalization is required, diuretics are often administeredintravenously because the ability to absorb oral diuretics may beimpaired. Side effects of diuretics include dehydration, electrolyteabnormalities, particularly low potassium levels, hearing disturbances,and low blood pressure. It is important to prevent low potassium levelsby providing supplements to patients, when appropriate. Any electrolyteimbalances may make patients susceptible to serious heart rhythmdisturbances. Examples of various classes of diuretics includefurosemide (LASIX), hydrochlorothiazide, bumetanide (BUMEX), torsemide(DEMADEX), and metolazone (ZAROXOLYN). Spironolactone (ALDACTONE) hasbeen used for many years as a relatively weak diuretic in the treatmentof various diseases. This drug blocks the action of the hormonealdosterone. Aldosterone has theoretical detrimental effects on theheart and circulation in congestive heart failure. Its release isstimulated in part by angiotensin II (supra). Side effects of this druginclude elevated potassium levels and, in males, breast tissue growth(gynecomastia). Another aldosterone inhibitor is eplerenone (INSPRA).

Relaxin Agonists

In some embodiments, the present disclosure provides methods of treatingdyspnea associated with acute heart failure in normotensive orhypertensive patients comprising administration of a relaxin agonist. Insome methods, the relaxin agonist activates one or more relaxin-relatedG-protein coupled receptors (GPCR) selected from but not limited toRXFP1, RXFP2, RXFP3, RXFP4, FSHR (LGR1), LHCGR (LGR2), TSHR (LGR3),LGR4, LGR5, LGR6 LGR7 (RXFP1) and LGR8 (RXFP2). In some embodiments, therelaxin agonist comprises the amino acid sequence of Formula I of WO2009/007848 of Compugen (herein incorporated by reference for theteaching of relaxin agonist sequences).

Formula I peptides are preferably from 7 to 100 amino acids in lengthand comprise the amino acid sequence:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33;wherein X1 is absent or G or a small naturally or non-naturallyoccurring amino acid; X2 is absent or Q or a polar naturally ornon-naturally occurring amino acid; X3 is absent or K or a basicnaturally or non-naturally occurring amino acid; X4 is absent or G or asmall naturally or non-naturally occurring amino acid; X5 is absent or Qor S a polar naturally or non-naturally occurring amino acid; X6 isabsent or V or A or P or M or a hydrophobic naturally or non-naturallyoccurring amino acid; X7 is absent or G or a small naturally ornon-naturally occurring amino acid; X8 is absent or P or L or Anaturally or non-naturally occurring amino acid; X9 is absent or P or Qnaturally or non-naturally occurring amino acid; X10 is absent or G or asmall naturally or non-naturally occurring amino acid; X11 is absent orA or H or E or D or a hydrophobic or a small or an acidic naturally ornon-naturally occurring amino acid; X12 is absent or A or P or Q or S orR or H or a hydrophobic or a small naturally or non-naturally occurringamino acid; X13 is absent or C or V or a hydrophobic naturally ornon-naturally occurring amino acid; X14 is absent or R or K or Q or P ora basic or a polar naturally or non-naturally occurring amino acid; X15is absent or R or Q or S or a basic or a polar naturally ornon-naturally occurring amino acid; X16 is absent or A or L or H or Q ora hydrophobic or a small naturally or non-naturally occurring aminoacid; X17 is absent or Y or a hydrophobic or an aromatic naturally ornon-naturally occurring amino acid; X18 is absent or A or a hydrophobicor small naturally or non-naturally occurring amino acid; X19 is absentor A or a hydrophobic small naturally or non-naturally occurring aminoacid; X20 is absent or F or a hydrophobic or an aromatic naturally ornon-naturally occurring amino acid; X21 is absent or S or T or a polarnaturally or non-naturally occurring amino acid; X22 is absent or V or ahydrophobic naturally or non-naturally occurring amino acid; X23 isabsent or G or hydrophobic or small non-naturally occurring amino acidor replaced by an amide; X24 is absent or R or a basic naturally ornon-naturally occurring amino acid; X25 is absent or R or a basicnaturally or non-naturally occurring amino acid; X26 is A or ahydrophobic or small naturally or non-naturally occurring amino acid;X27 is Y or a hydrophobic or an aromatic naturally or non-naturallyoccurring amino acid; X28 is A or a hydrophobic or small naturally ornon-naturally occurring amino acid; X29 is A or a hydrophobic or smallnaturally or non-naturally occurring amino acid; X30 is F or ahydrophobic naturally or non-naturally occurring amino acid; X31 is S orT or a polar naturally or non-naturally occurring amino acid; X32 is Vor a hydrophobic naturally or non-naturally occurring amino acid; X33 isabsent or G or hydrophobic or small naturally or non-naturally occurringamino acid or replaced by an amide; or a pharmaceutically acceptablesalt thereof (SEQ ID NO:4). In some preferred embodiments, the relaxinagonist comprises the sequence of peptide P59C13V (free acid)GQKGQVGPPGAA VRRA Y AAFSV (SEQ ID NO:5). In another preferredembodiment, the relaxin agonist comprises the sequence of peptideP74C13V (free acid) GQKGQVGPPGAA VRRA Y AAFS VGRRA Y AAFS V (SEQ DD NO:6). Further derivatives of the human complement C1Q tumor necrosisfactor-related protein 8 (CTRP8 or C1QT8) such as peptide P59-G (freeacid Gly) GQKGQVGPPGAACRRA Y AAFSVG (SEQ ID NO:7) are also contemplatedto be suitable for use in the methods of the present disclosure. Theamino acid sequence of C1QT8 is set forth as SEQ ID NO:8MAAPALLLLALLLPVGAWPGLPRRPCVHCCRPAWPPGPYARVSDRDLWRGDLWRGLPRVRPTIDIEILKGEKGEAGVRGRAGRSGKEGPPGARGLQGRRGQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVGRREGLHSSDHFQAVPFDTELVNLDGAFDLAAGRFLCTVPGVYFLSLNVHTWNYKETYLHIMLNRRPAAVLYAQPSERSVMQAQSLMLLLAAGDAVWVR MFQRDRDNAIYGEHGDLYITFSGHLVKP AAEL.

The present disclosure also encompasses encompasses homologues of thesepolypeptides, such homologues can be at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 85%, at least 90%, at least 95% or more say 100%identical to the amino acid sequence of an exemplary relaxin agonist(e.g., SEQ ID NO:5 or SEQ ID NO:6), as can be determined using BlastPsoftware of the National Center of Biotechnology Information (NCBI)using default parameters, optionally and preferably including thefollowing: filtering on (this option filters repetitive orlow-complexity sequences from the query using the Seg (protein)program), scoring matrix is BLOSUM62 for proteins, word size is 3, Evalue is 10, gap costs are 11, 1 (initialization and (initialization andextension). Optionally and preferably, nucleic acid sequenceidentity/homology is determined with BlastN software of the NationalCenter of Biotechnology Information (NCBI) using default parameters,which preferably include using the DUST filter program, and alsopreferably include having an E value of 10, filtering low complexitysequences and a word size of 11. Finally the present disclosure alsoencompasses fragments of the above described polypeptides andpolypeptides having mutations, such as deletions, insertions orsubstitutions of one or more amino acids, either naturally occurring orartificially induced, either randomly or in a targeted fashion.

Medical Uses

The disclosure provides medical uses of relaxin as defined above. Thus,for example, the disclosure provides a relaxin for use in treatingdyspnea in a human subject. In another embodiment the disclosureprovides a relaxin for use in treating acute decompensated heart failurein a human subject, wherein the subject has acute decompensated heartfailure and a systolic blood pressure of at least 125 mm Hg, and whereinthe method comprises administering the H2 relaxin to the subject in anamount effective to reduce their in hospital worsening heart failure. Inanother embodiment the disclosure provides a relaxin for use in treatingacute decompensated heart failure in a human subject, wherein thesubject has acute decompensated heart failure and a left ventricularejection fraction of at least about 20%, and wherein the methodcomprises administering the H2 relaxin to the subject in an amounteffective to reduce at least one acute heart failure sign or symptom inthe subject. The disclosure also provides a relaxin for use in treatingacute decompensated heart failure in a human subject, wherein thesubject has acute decompensated heart failure, and wherein the methodcomprises administering the H2 relaxin to the subject in an amounteffective to reduce diuretic use during a hospital stay.

The disclosure also provides the use of a relaxin in the manufacture ofa medicament for treating dyspnea in a human subject. The disclosurealso provides the use of a relaxin in the manufacture of a medicamentfor treating acute decompensated heart failure in a human subject,wherein the subject has acute decompensated heart failure and a systolicblood pressure of at least 125 mm Hg. The disclosure also provides theuse of a relaxin in the manufacture of a medicament for treating acutedecompensated heart failure in a human subject, wherein the subject hasacute decompensated heart failure and a left ventricular ejectionfraction of at least about 20%.

Other features of the relaxin and the treatments associated with theseuses are disclosed above.

The disclosure also provides the use of a relaxin and anantihypertensive drug in the manufacture of a medicament for treatingthe conditions discussed above. The antihypertensive drug may beselected as described above e.g. from the group consisting ofvasodilators, adrenergic blockers, centrally acting alpha-agonists,angiotensin-converting enzyme inhibitors, angiotensin II receptorblockers, calcium channel blockers and diuretics.

The disclosure also provides a relaxin and an antihypertensive drug, asa combined preparation for simultaneous separate or sequential use intreating the conditions discussed above. Similarly, the disclosureprovides a relaxin and an antihypertensive drug, for combined use intreating the conditions discussed above.

The disclosure also provides a relaxin for use, in combination with anantihypertensive drug, in treating the conditions discussed above.Similarly, the disclosure provides an antihypertensive drug for use, incombination with a relaxin, in treating the conditions discussed above.

The disclosure also provides a relaxin for use in a method for treatingthe conditions discussed above, wherein the relaxin is administered, oris prepared for administration, with an antihypertensive drug.Similarly, the disclosure provides an antihypertensive drug for use in amethod for treating the conditions discussed above, wherein theantihypertensive drug is administered, or is prepared foradministration, with a relaxin. The relaxin and/or antihypertensive drugmay also be used in this way in the manufacture of a medicament.

The disclosure also provides a relaxin for use in a method for treatingthe conditions discussed above, wherein the subject previously receivedan antihypertensive drug in the preceding 48 hours. Similarly, thedisclosure provides an antihypertensive drug for use in a method fortreating the conditions discussed above, wherein the subject previouslyreceived a relaxin drug in the preceding 48 hours. The relaxin and/orantihypertensive drug may also be used in this way in the manufacture ofa medicament. For these embodiments, the subjects may have received theother drug less than 48 hours previously e.g. in the preceding 24 hours,the preceding 12 hours, or the preceding 6 hours. Typically, thepreviously-administered drug will still be present in the subject's bodyand will be detectable. The remaining presence of thispreviously-administered drug distinguishes these subjects from thegeneral human population.

EXPERIMENTAL

The following specific examples are intended to illustrate thedisclosure and should not be construed as limiting the scope of theclaims.

Abbreviations: AHF (acute heart failure or decompensated congestiveheart failure); AUC (area under the curve); BNP (brain natriureticpeptide); (BP) blood pressure; BUN (blood urea nitrogen); CHF(congestive heart failure); CI (cardiac index); CO (cardiac output);CrCl (creatine clearance); DBP (diastolic blood pressure); dL(deciliters); eGFR (estimated glomerular filtration rate); hr (hour); HR(heart rate); ICU (intensive care unit); IV (intravenous); IVCD(intraventricular conduction delay); kg (kilogram); L (liter); LAHB(left anterior hemiblock); LBBB (left bundle branch block); LVEDP (leftventricular end diastolic pressure); LVEF (left ventricular ejectionfraction); mcg or μg(microgram); mEq (milliequivalents); MI (myocardialinfarction); mIU (milli-international units); mL (milliliter); NYHA (NewYork Heart Association); PAH (para-aminohippurate); PAP (pulmonaryarterial pressure); PCWP (pulmonary capillary wedge pressure); PD(pharmacodynamic); RAP (right atrial pressure); RBBB (right bundlebranch block); RBF (renal blood flow); rhRlx or rhRLX (recombinant humanrelaxin); Rlx or RLX (relaxin); RR (respiratory rate); SBP (systolicblood pressure); SI (stroke index); sMDRD (simplified Modification ofDiet in Renal Disease); SQ (subcutaneous SQ); SVR (systemic vascularresistance); T (temperature); VAS (visual analog scale); VF (ventricularfibrillation); VT (ventricular tachycardia); and WHF (worsening heartfailure).

Example 1 Study of Recombinant Human Relaxin in Patients with SystemicSclerosis

Overview. Clinical trials with relaxin have also been conducted onsystemic sclerosis patients. 257 human subjects who suffer from systemicsclerosis, a serious fibrotic disease, have been treated with relaxin bycontinuous and subcutaneous (SQ) infusion for six months. The results,which include extensive and long term safety information, have shownthat these patients did not experience any serious hypotensive events asa result of relaxin (FIG. 4), confirming the later CHF findings. Thesystemic sclerosis trials showed that relaxin administration wasassociated with stable decreases in blood pressure, with no seriousepisodes of hypotension, and a statistically significant increase inpredicted creatinine clearance (see FIG. 5). These findings support thehypothesis that relaxin administration was associated with balancedsystemic and renal vasodilation.

In addition, 570 human subjects have been treated with relaxin in 19completed trials. These subjects included patients with fibromyalgia,women undergoing egg donation, pregnant women at term, healthy femaleand male volunteers, healthy adults undergoing orthodontic therapy, andsystemic sclerosis patients.

Findings and Conclusion. As described herein, relaxin can beadministered safely in subjects with a variety of underlying conditions.In a number of these trials, data suggested that relaxin causes balancedsystemic and renal vasodilation.

Example 2 Study of Recombinant Human Relaxin in Patients with AcuteHeart Failure

Overview. A multi-center, randomized, double-blind, placebo-controlledclinical trial was conducted to determine the safety and efficacy ofrecombinant human relaxin (rhRLX) in patients with decompensatedcongestive heart failure (CHF). The terms decompensated CHF and acuteheart failure (AHF) are used interchangeably herein. Patientshospitalized for AHF (defined as including all of dyspnea at rest orwith minimal exertion, pulmonary congestion as evidenced by interstitialedema on chest radiograph, and an elevated BNP or NTproBNP), and havingan estimated glomerular filtration rate of 30-75 ml/min/1.73 m² and aSBP>125 mmHg at the time of screening were eligible for randomizationwithin 16 hours from presentation to standard AHF care plus a 48-hour IVinfusion of placebo or relaxin (RLX; 10, 30, 100 or 250 mcg/kg/d) andwere followed up to day 180. A total of 234 patients were enrolled inthe study.

Inclusion Criteria. Men and women aged 18 years or older who werehospitalized for AHF, with preserved or elevated blood pressure and withimpaired renal function were eligible for inclusion in the study. AHFwas defined by the presence of all of the following at screening:dyspnea at rest or with minimal exertion, pulmonary congestion on chestX-ray and elevated natriuretic peptide levels [brain natriuretic peptide(BNP)≧350 pg/mL or NT-pro-BNP≧1400 pg/mL]. Systolic blood pressure (SBP)had to be >125 mmHg at the time of screening. Impaired renal functionwas defined as an estimated glomerular filtration rate (eGFR) of between30 to 75 mL/min/1.73 m², calculated using the simplified Modification ofDiet in Renal Disease (sMDRD) equation (Levey et al., Ann Intern Med,130:461-470, 1999). Randomization was to occur within 16 hours ofinitial presentation. Patients had to qualify after receipt of at least40 mg of intravenous (IV) furosemide (or equivalent dose of alternativeloop diuretic).

Exclusion Criteria. Fever (temperature greater than 38° C.); acutecontrast-induced nephropathy or recent administration of contrast;ongoing or planned IV treatment with positive inotropic agents,vasopressors, vasodilators (with the exception of IV nitrates infused ata dose≦0.1 mg/kg/h if SBP>150 mmHg), or mechanical support (intra-aorticballoon pump, endotracheal intubation, mechanical ventilation or anyventricular assist device); severe pulmonary disease; significantstenotic cardiac valvular disease; previous organ transplantation oradmission for cardiac transplantation; clinical diagnosis of acutecoronary syndrome within 45 days prior to screening; major surgerywithin 30 days of screening; hematocrit less than 25%; major neurologicevent within 45 days prior to screening; troponin level at screeninggreater than 3 times the upper limit of normal; AHF caused bysignificant arrhythmias; non-cardiac pulmonary edema; or knownsignificant liver disease.

Study Drug. Recombinant human relaxin (rhRlx) was produced using aproprietary process as a single chain precursor, termedMini-C-prorelaxin, in a recombinant E. coli strain. Inclusion bodiescontaining the precursor were released from the cells by homogenizationand recovered by centrifugation. Mini-C-prorelaxin was extracted fromthe inclusion bodies, refolded with a redox buffer (in order to buildthe disulfide bridges), and partially purified by silica adsorption andion exchange chromatography. The leader sequence and the peptideconnecting the B-chain to the A-chain were then removed enzymatically.The resulting relaxin was then purified by three successivechromatography steps (ion exchange and reversed phase). Formulation ofthe product was achieved by ultra- and diafiltration. The rhRlx wasformulated as a sterile acetate buffered parenteral solution.

Study Procedures. The study was approved by the relevant ethicscommittees, institutional review boards and regulatory authorities, andconducted under the International Conference on Harmonization GoodClinical Practice guidelines. All patients provided informed writtenconsent prior to participation. Consenting patients who met all studyinclusion and none of the study exclusion criteria were randomized toreceive in double blind manner, either IV placebo or relaxin at 10, 30,100 or 250 mcg/kg/d for 48 hours in addition to standard therapy for AHFat the discretion of the investigator. The placebo used for the studywas the same solution as the diluent used to prepare the 100 μg/kg/daydose. The randomization ratio was 3:2:2:2:2, respectively. Relaxin(Corthera, San Mateo, Calif.) was manufactured using recombinanttechniques and was identical to the naturally-occurring peptide hormone.By protocol, the study drug infusion was to be terminated if thepatient's SBP was reduced to <100 mmHg or by >40 mmHg compared tobaseline in two successive measurements, 15 minutes apart. Investigatorswere not prohibited from utilizing any standard medication thoughtnecessary to treat patients enrolled in the study, including additionalvasodilators. Following a 4-hour washout period during which time IVvasodilators, IV pure inotropes and meals were withheld, hemodynamic,renal, and clinical responses to 48 hours of study drug infusion wereassessed.

Patient-reported dyspnea was assessed using both a standard 7-pointLikert Scale and a standard 100-mm Visual Analog Scale (VAS).Assessments were performed at baseline (VAS only), 6 h, 12 h, 24 h, 48 hafter initiation of drug therapy and at Days 3, 4, 5 and 14.Questionnaires were administered in the local language, andinvestigators received training in the standardized administration ofthese evaluations. Daily, serial physician-reported assessments of heartfailure signs and symptoms were conducted including jugular venousdistension, rales, edema, orthopnea, and dyspnea on exertion.In-hospital worsening heart failure was defined as aphysician-determined assessment based on worsening symptoms or signs ofheart failure and the need for the addition or institution of IVmedications or mechanical support to treat AHF. Vital status andrehospitalization information was collected by telephone at Day 30, Day60 and (vital status only at) Day 180. When the last enrolled patientreached Day 60, telephone contact was made with all patients who werebetween Day 60 and Day 180 of follow-up to complete the study.

Study Endpoints. As an exploratory, dose-finding study, Pre-RELAX-AHFdid not have a single pre-specified primary endpoint. Instead, theoverall effect of IV relaxin on seven primary treatment efficacy targetswas evaluated. 1.) Relief of dyspnea, assessed with two complementaryinstruments: (a) Change in dyspnea by Likert scale, and (b) Change frombaseline by Visual Analog Scale. 2.) In-hospital worsening heart failure(WHF) to Day 5. 3.) Renal impairment, assessed by multiple measures,including: (a) Renal impairment as defined by a ≧25% increase in serumcreatinine from baseline to day 5, and (b) Persistent renal impairmentas defined by creatinine increase of 0.3 mg/dL or above at both day 5and 14 from randomization. 4.) Length of initial hospital stay. 5.) Daysalive and out-of-hospital to Day 60. 6.) Death due to cardiovascularcauses or rehospitalization for heart failure or renal failure to Day60. 7.) Mortality due to cardiovascular causes to Day 180. In addition,serial assessments of safety were performed including vital signs,physical examinations, adverse events and clinical laboratoryevaluations.

Statistical Methods. Data are presented as means with standarddeviations unless otherwise specified. Missing data were generallyimputed by a last-observation-carried-forward approach. The worstobserved dyspnea Likert or VAS score was carried forward from the timeof death or worsening heart failure. The area under the curverepresenting the change in VAS score from baseline through Day 5 wascomputed by trapezoidal rule. For patients who died during the initialhospitalization, length of stay was imputed as the maximum observed plus1 day (33 days). Each relaxin group was compared to placebo, withoutadjustment for multiple comparisons, using logistic regression for thebinary outcomes, and the Wilcoxon rank sum test for continuous measures(with the van Elteren extension for the analysis of the length of stayand days alive out of hospital at Day 60), unless otherwise noted. Tocontrol for regional variations in this relatively small study, regionas a covariate or stratifying variable was prospectively pre-specifiedin the analyses of treatment effect. Rehospitalization and mortalityrates through Day 180 were estimated using Kaplan-Meier (product-limit)methods, and groups compared using the Wald test of the treatment effectfrom Cox regression models, where time-to-event was censored at lastpatient contact for patients without the event of interest.

The sample size in this phase 2 study was selected empirically and thestudy was not prospectively powered for statistical significance of anyspecific outcome measure. A p<0.05 was considered statisticallysignificant, while 0.05≦p≦0.20 was considered a trend suggestive of drugeffect. The main goals of the study were to identify a dose of relaxinthat was associated with multiple trends in the above mentioned primarytreatment targets and is not associated with safety concerns, todetermine which endpoints demonstrated treatment sensitivity and todocument the effect size for further statistical power calculations. Thechairperson of an unblinded, independent Data Safety and MonitoringBoard reviewed safety data monthly during the conduct of the study.

Study Population. The study enrolled 234 patients at 54 sites in 8countries (USA, Belgium, Italy, Poland, Israel, Hungary, Romania andRussia) from December, 2007 to August, 2008 with the final study contactin October, 2008. The safety analysis population consists of 230patients who received any amount of study drug. The efficacy analysispopulation consists of 229 patients who received study drug, excludingone patient who violated multiple major eligibility criteria. Patientswere 70.3±10.5 years old and 56% male, with a screening blood pressureof 147±19 mmHg and extensive co-morbidities (Table 3-1). There were noclinically meaningful or statistically significant differences incharacteristics among the five treatment groups. Patients wererandomized at a mean of 8.4±5.4 hours from presentation [median 6.6hours (Q1-Q3: 4.0-13.4)] and were treated with study drug within 1.0±1.8hours from randomization. Patients in the placebo group received a meanduration of infusion of 44 hours, while those in the relaxin 10, 30, 100and 250 mcg/kg/d groups received an average of 39, 41, 41 and 42 hoursof study drug, respectively. Patients received standard therapy inaddition to study drug with 18.0% of the placebo group receivingintravenous nitroglycerin during the first 24 hours, compared to 10.0%,9.5%, 13.5 and 4.1% in the relaxin 10, 30, 100 and 250 mcg/kg/d groups,respectively.

Dyspnea Responses. Results are presented via the Visual Analog Score(VAS) and the Likert Score. The VAS score measures a characteristic orattitude that ranges across a continuum of values. For example, theamount of discomfort an AHF patient feels ranges across a continuum fromnone to an extreme amount of discomfort and/or pain including dyspnea,hypertension, high blood pressure, arrhythmia and reduced renal bloodflow. From the patient's perspective this spectrum appears continuous,which the VAS captures. Operationally a VAS is usually a horizontalline, 100 mm in length, anchored by word descriptors at each end (e.g.,no discomfort on one end and severe discomfort on the other end). Thepatients marked on the line the point that they felt represented theirperception of their current state. The VAS score is determined bymeasuring in millimeters from the left hand end of the line to the pointthat the patient marks (Wewers et al., Research in Nursing and Health13:227-236, 1990).

The Likert Score is a unidimensional scaling method known in the art,wherein the set of scale items are rated on a numerical (herein 7-point)Disagree-Agree response scale. Each patient was asked to rate each itemon the response scale. The final score for the respondents on the scaleis the sum of their ratings for all of the items.

Relaxin-treated patients had rapid, meaningful and sustained dyspneaimprovement compared to those in the placebo group. The combinedrelaxin-treated group had a larger improvement in dyspnea severitycompared to placebo as early as 6 hours after initiation of therapy,persisting throughout all time points assessed. The best response totreatment was observed in the patients receiving relaxin at the dose of30 mcg/kg/d. Moderately or markedly better dyspnea on the Likert Scaleat all of the 6 h, 12 h and 24 h assessments occurred in 23.0% ofpatients in the placebo group compared to 40.5% in the relaxin 30mcg/kg/d group (p=0.044; Table 3-2). The VAS similarly demonstrated asustained, positive trend of drug effect on relief of dyspnea. The areaunder the curve (AUC) for change from baseline to Day 5 in the dyspneaVAS was 1679±2556 mm*hr in the placebo group compared to 2567±2898mm*hour in the relaxin 30 mcg/kg/d group (p=0.11; Table 3-2), and theseobserved changes correspond to averages of 14, 21, 22, 21 and 18 mmimprovement over the 5 days for the placebo and relaxin 10, 30, 100 and250 mcg/kg/d groups, respectively. Similar results are evident for theVAS AUC through Day 14 (Table 3-2) where placebo mean was 4622±9003mm*hr compared to 8214±8712 mm*hour in the relaxin 30 mcg/kg/d group(p=0.053). These changes correspond to averages of 14, 10, 25, 25 and 21mm over the 14 days, for the respective groups.

Short-term Outcomes. There were consistent trends (p<0.20) in favor ofrelaxin therapy compared to placebo in multiple in-hospital assessments.In particular, the relaxin dose of 30 mcg/kg/d appeared most effectivewith supportive trends in the groups receiving 10 and 100 mcg/kg/d.Physician-assessed resolution of jugular venous distension, rales, andedema were all improved in the relaxin 30 mcg/kg/d group compared toplacebo at Day 5 (Table 3-3) and at Day 14, associated with trendstoward greater decrease in body weight and decreased diuretic use in therelaxin-treated patients. The cumulative incidence of worsening heartfailure by day 5 was lower in the relaxin groups compared to placebo(Table 3-2), and the mean length of stay for the index hospitalizationtended to be 0.9-1.8 days shorter in the relaxin groups than for placebo(Table 3-2; p=0.18 for relaxin 30 mcg/kg/d vs. placebo group).

Post-Discharge Outcomes. Patients were followed for an average of 122±53days. A total of 15 patients died by Day 60, and 20 patients by Day 180,12 for cardiovascular causes. Forty-three patients were rehospitalizedby Day 60; 15 due to heart failure and none due to renal failure.Relaxin-treated patients demonstrated trends toward improvement inlonger-term clinical outcomes (Table 3-2). At Day 60, the mean number ofdays alive and out-of-hospital was 44.2±14.2 in the placebo group, whileit was approximately 4 days greater in the relaxin-treated patients(p=0.16 for 30 mcg/kg/d vs. placebo group). The Kaplan-Meier estimate ofthe combined incidence of death due to cardiovascular causes orrehospitalization due to heart failure or renal failure at day 60 was17.2% in the group receiving placebo, but much less in therelaxin-treated patients with an estimated 87% hazard reduction in therelaxin 30 mcg/kg/d group (p=0.053 vs. placebo). Similar findings wereevident when all-cause mortality was included (Table 3-2). TheKaplan-Meier estimate of Day 180 cardiovascular mortality was 14.3% inthe placebo group, but was considerably less in the relaxin-treatedgroups (p=0.046 for relaxin 30 mcg/kg/d compared to placebo by Fisher'sexact test of the incidence densities). The corresponding Kaplan-Meierestimates for all-cause mortality demonstrated similar trends.

Safety Endpoints. Adverse events and serious adverse events were evenlydistributed across study groups and represented the natural history ofpatients hospitalized with AHF (Table 3-4). There were no individual orpattern of adverse events suggesting a deleterious study drug effect.

Relaxin has known vasodilating activity and consequently, changes inblood pressure were carefully monitored. During the 48-hour infusionperiod, the placebo group had a 12-20 mmHg decrease from baseline insystolic blood pressure (SBP) and the relaxin-treated patients hadsimilar reductions (FIG. 21). The average decrease in blood pressureover all time points did not differ between any of the treatment groupsand the placebo group by repeated measures ANOVA (p-values for theaverage change in SBP comparing 10, 30, 100, and 250 mcg/kg/day withplacebo were 0.41, 0.16, 0.13, and 0.32, respectively), although therewas a trend in the 30 and 100 mcg/kg/d groups with a mean decrease of3-4 mmHg compared to placebo. There were 36 adverse events ofhypotension and/or decreases in SBP which met protocol-specified studydrug stopping rules, two of which were serious adverse events (both inthe relaxin 250 mcg/kg/d group). Protocol-specified study drugdiscontinuation due to blood pressure reduction occurred in 10.9% ofpatients across all groups, and was more frequent in relaxin-treatedgroups (20.0%, 9.5%, 7.9% and 16.3% with relaxin 10, 30, 100 and 250mcg/kg/d, respectively) compared to placebo (3.3%) with no apparentdose-response. Most blood pressure reductions occurred during the first6-12 hours of therapy. In no cases did the trough SBP fall below 80mmHg. After discontinuation of study drug, SBP stabilized or rose inmost of these patients with no therapy (1 of 2 placebo patients with SBPreductions; 18 of 23 relaxin-treated patients). In the placebo group, 1patient (1.6%) received intravenous fluids for hypotension, while 5patients from the four relaxin-treated groups (3.0%) receivedintravenous fluids and one asymptomatic patient also received dobutaminein the relaxin 250 mcg/kg/d group. None of the patients in the 10 or 30mcg/kg/d groups required treatment of blood pressure reduction.

There were no differences in the incidence of renal failure reported asa serious adverse event among the study groups (Table 3-4). At Day 14,mean changes in creatinine from baseline were 0.08±0.46, 0.07±0.24,0.13±0.49, 0.08±0.39 and 0.10±0.39 mg/dL (p-value for each group vs.placebo≧0.97). The proportion of patients at Day 14 with an increase of0.3 mg/dL or more was 16.7%, 19.4%, 26.3%, 24.2% and 37.2% (p=0.03 for250 mcg/kg/d vs. placebo). Persistent renal impairment (0.3 mg/dL orgreater increase in creatinine at both Day 5 and 14) also trended tobeing greater in patients receiving relaxin 250 mcg/kg/d (p=0.19 vs.placebo).

As with many vasodilators, there was a transient and clinicallyinsignificant decrease in hematocrit in all active treatment groups thatoccurred during study drug administration (change from baseline in meanhematocrit at 48 hours: +0.42% in placebo group and 0.57%, 1.45%, 0.25%,0.64% in relaxin 10, 30, 100 and 250 mcg/kg/d groups, respectively;p=0.019 vs. placebo for relaxin 30 mcg/kg/d group), resolving by Day 5.There were no other clinical laboratory changes of note during thestudy.

TABLE 3-1 Baseline Patient Characteristics Relaxin (mcg/kg/d) GroupPlacebo 10 30 100 250 Number of Subjects in 61 40 42 37 49 EfficacyAnalysis Men, % 65.6 52.5 42.9 51.4 61.2 Age, yr 68.4 (9.9)  72.2 (11.0)71.6 (9.2)  69.2 (11.6) 70.7 (11.0) Weight, kg 80.7 (15.6) 80.2 (16.9)79.9 (13.0) 84.5 (25.0) 80.2 (16.7) Ischemic heart disease, % 67.2 62.578.6 64.9 73.5 Hypertension history, % 82.0 87.5 90.5 81.1 87.8 Diabeteshistory, % 49.2 32.5 52.4 32.4 40.8 Mitral regurgitation, % 23.0 30.031.0 32.4 36.7 Atrial fibrillation/flutter, % 42.6 60.0 42.9 56.8 38.8Ejection fraction <40%, % 44.2 48.4 53.6 68.0 55.6 Hospitalized for AHFin 29.5 32.5 38.1 43.2 30.6 prior year, % NYHA class, % I 3.3 0.0 0.00.0 4.1 II 26.2 35.0 14.3 21.6 10.2 III 37.7 42.5 40.5 35.1 44.9 IV 19.712.5 33.3 37.8 28.6 NT-pro-BNP 75.4 70.0 83.3 70.3 71.4 >2000 pg/mL, %Troponin ≧0.1 ng/mL and 23.3 18.4 10.3 13.9 16.7 <3 × ULN, % SBP atscreening, mmHg 147.5 (20.3)  145.4 (16.0)  150.3 (19.5)  146.5 (18.7) 145.5 (20.5)  eGFR 53.9 (16.8) 56.5 (15.8) 50.6 (14.1) 53.4 (22.0) 53.4(15.2) Serum creatinine, mg/dL 1.4 (0.5) 1.2 (0.5) 1.3 (0.4) 1.3 (0.4)1.3 (0.5) BUN, mg/dL 28.3 (12.4) 25.2 (11.7) 28.2 (10.7) 25.7 (10.7)26.7 (10.8) Sodium, meq/L 140.7 (3.4)  139.9 (3.2)  140.4 (4.0)  140.8(4.1)  139.9 (4.9)  Time from presentation to 9.0 (5.7) 7.5 (4.8) 7.6(4.8) 9.0 (5.5) 8.4 (5.7) randomization, hr [median] [6.4] [6.0] [6.1][7.5] [6.6] Time from randomization to 1.0 (1.1) 0.9 (1.2) 0.6 (0.5) 0.7(0.4) 1.6 (3.6) drug administration, hr Medications 1 month prior topresentation, % ACE inhibitor or ARB 75.4 55.0 73.8 75.7 69.4Beta-blocker 60.7 67.5 69.0 59.5 63.3 Aldosterone inhibitor 27.9 27.528.6 29.7 38.8 Results expressed as mean (SD), unless otherwise noted.NYHA (New York Heart Association) class when last in stable condition;eGFR by sMDRD, ml/min/1.73 m²; ULN, upper limit of normal.

TABLE 3-2 Effect Of Relaxin On Primary Treatment Targets Relaxin(mcg/kg/d) Placebo 10 30 100 250 Number of Subjects in 61 40 42 37 49Efficacy Analysis Short-term Outcomes: % moderately/markedly 23.0% 27.5%40.5% 13.5% 22.4% better dyspnea at 6, 12 p = 0.54  p = 0.044 p = 0.28 p= 0.86 and 24 hrs (Likert) Dyspnea AUC Change 1679 ± 2556 2500 ± 29082567 ± 2898 2486 ± 2865 2155 ± 2338 from baseline to Day 5 p = 0.15 p =0.11 p = 0.16 p = 0.31 (VAS; mm * hr) Dyspnea AUC Change 4621 ± 9003 6366 ± 10078 8214 ± 8712 8227 ± 9707 6856 ± 7923 from baseline to Day14 p = 0.37  p = 0.053  p = 0.064 p = 0.16 (VAS; mm * hr) Worsening HFthrough 21.3% 20.0% 11.9% 13.5% 10.2% Day 5 (%)* p = 0.75 p = 0.29 p =0.40 p = 0.15 Length of Hospital Stay 12.0 ± 7.3  10.9 ± 8.5  10.2 ±6.1  11.1 ± 6.6  10.6 ± 6.6  (days) p = 0.36 p = 0.18 p = 0.75 p = 0.20Day 60 Outcomes Days alive out of hospital 44.2 ± 14.2 47.0 ± 13.0 47.9± 10.1   48 ± 10.1 47.6 ± 12.0 p = 0.40 p = 0.16 p = 0.40  p = 0.048Cardiovascular death or 17.2% 10.1% 2.6% 8.4% 6.2% Rehospitalization (KM%; [0.55 (0.17-1.77)] [0.13 (0.02-1.03)] [0.46 (0.13-1.66)] [0.32(0.09-1.17)] [HR (95% CI)])† p = 0.32 p = 0.053 p = 0.23  p = 0.085All-cause death or 18.6% 12.5% 7.6% 10.9% 8.3% Rehospitalization (KM %;[0.63 (0.22-1.81)] [0.36 (0.10-1.29)] [0.56 (0.18-1.76)] [0.41(0.13-1.28)] [HR (95% CI)])† p = 0.39 p = 0.12 p = 0.32 p = 0.12 Day 180Outcomes Cardiovascular death 14.3% 2.5% 0.0% 2.9% 6.2% (KM %; [HR (95%[0.19 (0.00-1.49)] [0.00 (0.00-0.98)] [0.23 (0.01-1.79)] [0.56(0.09-2.47)] CI)])**, † p = 0.15  p = 0.046 p = 0.17 p = 0.53 All-causedeath (KM %; 15.8% 5.0% 8.7% 5.5% 10.7% [HR (95% CI)])† [0.34(0.07-1.62] [0.54 (0.14-2.03] [0.41 (0.09-1.91] [0.08 (0.26-2.47] p =0.18 p = 0.36 p = 0.25 p = 0.70 Results expressed as mean ± SD; *ForWilcoxon rank sum test of time to worsening HF through Day 5; subjectswithout worsening HF were assigned a value of 6 days; **by Fisher'sexact test comparing incidence densities; †Analyses performed on safetypopulation which included one additional patient (n = 38) in the 100mcg/kg/d group. Rehospitalization included hospitalization for heartfailure or renal failure; KM, Kaplan-Meier estimates of event rate atspecified time period; HR, hazard ratio.

TABLE 3-3 Improvement in Signs of Heart Failure Relaxin (mcg/kg/d)Placebo 10 30 100 250 Number of Subjects in 61 40 42 37   49 Efficacypopulation % of subjects at Day 5 with: No edema 47.5 55.0 64.3†  51.4*61.2† No rales 67.2 65.0 76.2 70.3 71.4 JVP <6 cm 67.2 72.5 78.6 73.076.6+ Median total IV diuretic dose 170 100 100 90+  140 fromrandomization to Day 5  (80-300)  (40-200)  (60-360)  (40-200)  (60-340)[mg; median (Q1-Q3)] Median change in body −2.0 −2.0 −3.0 −2.5 −2.0weight from baseline to Day (−4.2-0.0)   (−4.5-0.0)   (−5.0-0.0)  (−4.7-0.8)   (−4.0-0.0)   14 [kg; median (Q1; Q3)] †p < 0.001; *0.001 ≦p ≦ 0.05; +0.05 < p ≦ 0.20 for Wilcoxon rank sum test of change in scorefrom baseline (for signs), van Elteren extension of the Wilcoxon test(for diuretic dose), or ANOVA (for body weight). JVP, jugular venouspressure.

TABLE 3-4 Selected Adverse Events Relaxin (mcg/kg/d) Group Placebo 10 30100 250 Number of Subjects in Safety 61 40 42 38 49 Groups Seriousadverse events (SAEs) to Day 30 Patients with any SAEs to Day 10 (16.4%) 7 (17.5%)  7 (16.7%) 3 (7.9%)  8 (16.3%) 30, n (%) Total number of SAEs13 8 12 3 11 Cardiac failure, n (%) 5 (8.2%) 2 (5.0%) 1 (2.4%) 0 2(6.1%) Ventricular fibrillation, n (%) 0 1 (2.5%) 0 0 0 Noncardiac chestpain, n (%) 0 2 (5.0%) 0 0 1 (2.0%) Hypotension, n (%) 0 0 0 0 2 (4.1%)Acute respiratory failure, n (%) 0 0 0 1 (2.6%) 0 Pneumonia, n (%) 1(1.6%) 0 3 (7.1%) 0 0 Bronchitis, n (%) 0 0 1 (2.4%) 0 1 (2.0%) Urinarytract infection, n (%) 0 0 1 (2.4%) 0 1 (2.0%) Cerebrovascular accident,n (%) 1 (1.6%) 0 2 (4.8%) 0 0 Renal failure, n (%) 1 (1.6%) 0 1 (2.4%) 00 Urinary retention, n (%) 0 0 0 2 (5.3%) 0 Adverse events to Day 30Patients with any adverse 45 (73.8%) 32 (80.0%) 25 (59.5%) 24 (63.2%) 25(51.0%) events to Day 30, n (%) Patients with any AE from Day 6 (9.8%) 4 (10.0%)  5 (11.9%) 2 (5.3%) 3 (6.1%) 15 to Day 30, n (%) Renalimpairment Patients with ≧25% increase in  8 (13.3%)  4 (10.0%)  9(22.0%)  11 (29.7%)* 12 (25.5%) creatinine at Day 5 Patients with ≧0.3mg/dL 11 (19.3%) 3 (7.9%)  7 (18.9%)  9 (26.5%) 10 (22.7%) increase increatinine at Day 5 Patients with ≧0.3 mg/dL 4 (6.8%) 3 (7.5%) 3 (7.3%) 4 (10.8%)    7 (15.2%)+ increase in creatinine at Day 5 and Day 14 *P <0.05; +p < 0.20.

Findings. As shown in FIGS. 6-11 of the interim analysis and FIGS. 12and 13 of the final analysis, relaxin treatment resulted in measurableimprovements in dyspnea. Although all patients received benefit fromrelaxin treatment, patients with NT-pro-BNP of greater than 2000,patients with systolic blood pressure greater than the median, andpatients with creatinine clearance of less than the median, received thegreatest benefit (FIGS. 7-11). Surprisingly, a low dosage of 30μg/kg/day of relaxin provided the most rapid and marked relief ofdyspnea as measured using a 7-point Likert score (FIG. 12). Across allrelaxin-treated groups, the trends in VAS measurements (FIG. 13) ofdyspnea also unexpectedly indicated that the beneficial effect ofrelaxin treatment was persistent (e.g., through day 14). Bothinstruments (VAS and Likert) are accepted measures of dyspnea in heartfailure patients, although the categorical scale (Likert) appears moresensitive to early changes, while the ordinal scale (VAS) appears moresensitive to late changes.

The beneficial effect of relaxin included a reduction of acute cardiacdecompensation events including not only dyspnea, but extra body weightdue to retention of fluids, length of hospital stay, likelihood ofhospital re-admission, need for loop diuretics, need for intravenous(IV) nitroglycerin, and an incidence of worsening heart failure (FIGS.14-19). Specifically a decrease in the incidence of worsening of heartfailure compared to placebo was found to be clinically relevant, whileshorter hospital stays and a reduced incidence of re-hospitalizationpromises a positive impact in pharma-economics. In addition, there wereno apparent adverse effects on renal function, and there were no safetyor tolerability issues. Noteworthy in their absences were untoward heartrate elevations and symptomatic hypotension in relaxin-treated patients(see, FIGS. 20 and 21), which one of skill in the art may have expectedof a chronotropic agent or an indiscriminate vasodilator.

Conclusion. This is the first prospective study to examine the effectsof IV relaxin in patients with acute heart failure (AHF), presentingwith systolic blood pressure greater than 125 mmHg and mild to moderaterenal impairment. Treatment with relaxin was associated with significantimprovement in dyspnea that was substantial in magnitude, rapid in onset(within 6 hours), and sustained to 14 days. Treatment with relaxin wasassociated with trends toward improvement in other important clinicalendpoints, including signs of heart failure, in-hospital worsening ofheart failure, length of stay, cardiovascular death or rehospitalizationat 60 days, and 180-day cardiovascular mortality. These effects weremost marked in the 30 mcg/kg/d relaxin group, although similar butsmaller trends were seen with 10 and 100 mcg/kg/d doses of relaxin.There were no concerning safety signals for relaxin in AHF patientsidentified in this study.

Various modifications and variations of the present disclosure will beapparent to those skilled in the art without departing from the scopeand spirit of the disclosure. Although the disclosure has been describedin connection with specific preferred embodiments, it should beunderstood that the claims should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the disclosure, which are understood by those skilled inthe art are intended to be within the scope of the claims.

1. A method for treating dyspnea associated with acute heart failure,comprising: administering to a human subject a pharmaceutically activeH2 relaxin in an amount effective to reduce dyspnea in said subject,wherein said subject has dyspnea associated with acute heart failure andis in a hypertensive or normotensive state at the onset of saidadministering.
 2. The method of claim 1, further comprising selectingsaid human subject having dyspnea associated with acute heart failureand in a hypertensive or normotensive state, prior to said administeringstep.
 3. The method of claim 2, wherein said H2 relaxin is administeredfor at least 24 hours or at least 48 hours.
 4. The method of claim 3,wherein said H2 relaxin is administered at an intravenous infusion ratein the range of about 10 μg/kg/day to about 250 μg/kg/day.
 5. The methodof claim 4, wherein said H2 relaxin is administered at an intravenousinfusion rate in the range of about 30 μg/kg/day to about 100 μg/kg/day.6. The method of claim 5, wherein said H2 relaxin is administered at anintravenous infusion rate of about 30 μg/kg/day.
 7. The method of claim6, wherein the reduction in dyspnea is statistically significant at 6hours after the onset of treatment compared to treatment without H2relaxin.
 8. The method of claim 6, wherein the reduction in dyspnea isstatistically significant at 12 hours after the onset of treatmentcompared to treatment without H2 relaxin.
 9. The method of claim 6,wherein the reduction in dyspnea is statistically significant at 6, 12and 24 hours after the onset of treatment compared to placebo.
 10. Themethod of claim 6, wherein the reduction in dyspnea lasts for at leastabout twice the duration of treatment.
 11. The method of claim 6,wherein the reduction in dyspnea lasts for at least about 4 times theduration of treatment.
 12. The method of claim 6, wherein the reductionin dyspnea lasts for at least about 7 times the duration of treatment.13. The method of claim 5, further comprising reducing the body weightof said subject by at least about 0.5 kg over a 14-day period comparedto treatment without H2 relaxin.
 14. The method of claim 6, furthercomprising reducing the body weight of said subject by at least about 1kg over a 14-day period compared to treatment without H2 relaxin. 15.The method of claim 1, wherein said subject is renally impaired.
 16. Themethod of claim 15, wherein said subject has a creatinine clearance inthe range of about 35 to about 75 mL/min.
 17. The method of claim 1,further comprising reducing the 60-day risk of death orrehospitalization of said subject compared to treatment of acutedecompensated heart failure without H2 relaxin.
 18. The method of claim17, wherein the 60-day risk of death or rehospitalization is reduced byat least 50%.
 19. The method of claim 1, wherein said subject hasdyspnea requiring hospitalization.
 20. The method of claim 19, furthercomprising reducing the hospitalization length of stay by at least oneday compared to treatment of acute decompensated heart failure withoutH2 relaxin.
 21. The method of claim 20, wherein said H2 relaxin isadministered at an intravenous infusion rate of about 30 μg/kg/day andthe hospitalization length of stay is reduced by at least two dayscompared to treatment of acute decompensated heart failure without H2relaxin.
 22. The method of claim 19, further comprising reducing the60-day risk of rehospitalization due to heart failure or renalinsufficiency of said subject compared to treatment of acutedecompensated heart failure without H2 relaxin.
 23. The method of claim22, wherein the 60-day risk of rehospitalization due to heart failure orrenal insufficiency is reduced by at least about 50%.
 24. The method ofclaim 23, wherein said H2 relaxin is administered at an intravenousinfusion rate of about 30 μg/kg/day and the 60-day risk ofrehospitalization due to heart failure or renal insufficiency is reducedby at least about 70%.
 25. The method of claim 1, further comprisingreducing the 180-day risk of cardiovascular death of said subjectcompared to treatment of acute decompensated heart failure without H2relaxin.
 26. The method of claim 25, wherein the 180-day risk ofcardiovascular death is reduced by at least about 50%.
 27. The method ofclaim 25, wherein said H2 relaxin is administered at an intravenousinfusion rate less than about 250 μg/kg/day and the 180-day risk ofcardiovascular death is reduced by at least about 70%.
 28. The method ofclaim 1, further comprising reducing the 180-day risk of all-causemortality of said subject compared to treatment of acute decompensatedheart failure without H2 relaxin.
 29. The method of claim 28, whereinthe 180-day risk of all-cause mortality is reduced by at least about25%.
 30. The method of claim 29, wherein said H2 relaxin is administeredat an intravenous infusion rate less than about 250 μg/kg/day and the180-day risk of all-cause mortality is reduced by at least about 50%.31. A method for treating dyspnea associated with acute decompensatedheart failure, comprising: administering to a human subject apharmaceutically active H2 relaxin in an amount effective to reducedyspnea in said subject, wherein said subject has dyspnea associatedwith acute decompensated heart failure and at least one indicia ofcardiac ischemia.
 32. The method of claim 31, further comprisingselecting said human subject having dyspnea associated with acutedecompensated heart failure and at least one indicia of cardiacischemia, prior to said administering step.
 33. The method of claim 31,where the at least one indicia of cardiac ischemia is selected from thegroup consisting of a positive troponin test, an abnormalelectrocardiogram, the presence of chest pain, the presence of anarrhythmia, a positive creatine kinase-MB test, and an abnormalechocardiogram.
 34. A method for treating dyspnea associated with acutedecompensated heart failure, comprising: administering to a humansubject a pharmaceutically active H2 relaxin in an amount effective toreduce dyspnea in said subject, wherein said subject has acutedecompensated heart failure and a left ventricular ejection fraction ofat least about 20%.
 35. The method of claim 34, wherein said subject hasa left ventricular ejection fraction in the range of about 20% to about40%.
 36. The method of claim 34, wherein said subject has a leftventricular ejection fraction of at least about 40%.
 37. A method fortreating acute decompensated heart failure, comprising: a) selecting asubject with acute decompensated heart failure and a systolic bloodpressure of at least 125 mm Hg; and b) administering to said subject apharmaceutically active H2 relaxin in an amount effective to reducein-hospital worsening heart failure in said subject.
 38. The method ofclaim 37, wherein said in-hospital worsening heart failure comprises oneor more of worsening dyspnea, need for additional intravenous therapy totreat said heart failure, need for mechanical support of breathing, andneed for mechanical support of blood pressure.
 39. The method of claim38, further comprising reducing the 60-day risk of death orrehospitalization of said subject compared to treatment of acutedecompensated heart failure without H2 relaxin.
 40. A method fortreating acute decompensated heart failure, comprising: a) selecting asubject with acute decompensated heart failure and a left ventricularejection fraction of at least about 20%; and b) administering to saidsubject a pharmaceutically active H2 relaxin in an amount effective toreduce at least one acute heart failure sign or symptom in said subject.41. The method of claim 40, wherein said at least one acute heartfailure sign or symptom comprises one or more of the group consisting ofdyspnea at rest, orthopnea, dyspnea on exertion, edema, rales, pulmonarycongestion, jugular venous pulse or distension, edema associated weightgain, high pulmonary capillary wedge pressure, high left ventricularend-diastolic pressure, high systemic vascular resistance, low cardiacoutput, low left ventricular ejection fraction, need for intravenousdiuretic therapy, need for additional intravenous vasodilator therapy,and incidence of worsening in-hospital heart failure.
 42. The method ofclaim 40, wherein said subject has a left ventricular ejection fractionof at least 40%.
 43. A method for treating acute decompensated heartfailure, comprising administering to a subject with acute decompensatedheart failure a pharmaceutically active H2 relaxin in an amounteffective to reduce diuretic use during a hospital stay compared totreatment of acute decompensated heart failure without using H2 relaxin.44. The method of claim 43, wherein said subject is renally impaired.45. The method of claim 44, wherein said subject has a creatinineclearance in the range of about 35 to about 75 mL/min.